Cellular Stress Response to Engineered Nanoparticles: Effect of Size, Surface Coating, and Cellular Uptake

EPA Science Inventory

CELLULAR STRESS RESPONSE TO ENGINEERED NANOPARTICLES: EFFECT OF SIZE, SURFACE COATING, AND CELLULAR UPTAKE RY Prasad 1, JK McGee2, MG Killius1 D Ackerman2, CF Blackman2 DM DeMarini2 , SO Simmons2 1 Student Services Contractor, US EPA, RTP, NC 2 US EPA, RTP, NC The num...

Enantioselective cellular uptake of chiral semiconductor nanocrystals

NASA Astrophysics Data System (ADS)

Martynenko, I. V.; Kuznetsova, V. A.; Litvinov, I. K.; Orlova, A. O.; Maslov, V. G.; Fedorov, A. V.; Dubavik, A.; Purcell-Milton, F.; Gun'ko, Yu K.; Baranov, A. V.

2016-02-01

The influence of the chirality of semiconductor nanocrystals, CdSe/ZnS quantum dots (QDs) capped with L- and D-cysteine, on the efficiency of their uptake by living Ehrlich Ascite carcinoma cells is studied by spectral- and time-resolved fluorescence microspectroscopy. We report an evident enantioselective process where cellular uptake of the L-Cys QDs is almost twice as effective as that of the D-Cys QDs. This finding paves the way for the creation of novel approaches to control the biological properties and behavior of nanomaterials in living cells.

FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel.

PubMed

Saari, H; Lisitsyna, E; Rautaniemi, K; Rojalin, T; Niemi, L; Nivaro, O; Laaksonen, T; Yliperttula, M; Vuorimaa-Laukkanen, E

2018-06-15

In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti-cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel-containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real-time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next-generation targeted drug delivery systems. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

On Guanidinium and Cellular Uptake

PubMed Central

2015-01-01

Guanidinium-rich scaffolds facilitate cellular translocation and delivery of bioactive cargos through biological barriers. Although impressive uptake has been demonstrated for nonoligomeric and nonpept(o)idic guanidinylated scaffolds in cell cultures and animal models, the fundamental understanding of these processes is lacking. Charge pairing and hydrogen bonding with cell surface counterparts have been proposed, but their exact role remains putative. The impact of the number and spatial relationships of the guanidinium groups on delivery and organelle/organ localization is yet to be established. PMID:25019333

The Role of Hydrophobicity in the Cellular Uptake of Negatively Charged Macromolecules.

PubMed

Abou Matar, Tamara; Karam, Pierre

2018-02-01

It is generally accepted that positively charged molecules are the gold standard to by-pass the negatively charged cell membrane. Here, it is shown that cellular uptake is also possible for polymers with negatively charged side chains and hydrophobic backbones. Specifically, poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene], a conjugated polyelectrolyte with sulfonate, as water-soluble functional groups, is shown to accumulate in the intracellular region. When the polymer hydrophobic backbone is dissolved using polyvinylpyrrolidone, an amphiphilic macromolecule, the cellular uptake is dramatically reduced. The report sheds light on the fine balance between negatively charged side groups and the hydrophobicity of polymers to either enhance or reduce cellular uptake. As a result, these findings will have important ramifications on the future design of targeted cellular delivery nanocarriers for imaging and therapeutic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

PubMed Central

2012-01-01

Background It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical and initial imaging analysis indicated that productive fusion events occur predominantly within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Quantitative monitoring of the fraction of individual viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes

Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

PubMed Central

2015-01-01

To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

PubMed

Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

2015-12-15

NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP. Copyright © 2015 Elsevier B.V. All rights reserved.

Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles.

PubMed

Sun, Dequan; Hussain, Hashmath I; Yi, Zhifeng; Siegele, Rainer; Cresswell, Tom; Kong, Lingxue; Cahill, David M

2014-08-01

We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry

PubMed Central

2013-01-01

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs. PMID:23388071

Preferential tumor cellular uptake and retention of indocyanine green for in vivo tumor imaging.

PubMed

Onda, Nobuhiko; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

2016-08-01

Indocyanine green (ICG) is a fluorescent agent approved for clinical applications by the Food and Drug Administration and European Medicines Agency. This study examined the mechanism of tumor imaging using intravenously administered ICG. The in vivo kinetics of intravenously administered ICG were determined in tumor xenografts using microscopic approaches that enabled both spatio-temporal and high-magnification analyses. The mechanism of ICG-based tumor imaging was examined at the cellular level in six phenotypically different human colon cancer cell lines exhibiting different grades of epithelioid organization. ICG fluorescence imaging detected xenograft tumors, even those < 1 mm in size, based on their preferential cellular uptake and retention of the dye following its rapid tissue-non-specific delivery, in contrast to its rapid clearance by normal tissue. Live-cell imaging revealed that cellular ICG uptake is temperature-dependent and occurs after ICG binding to the cellular membrane, a pattern suggesting endocytic uptake as the mechanism. Cellular ICG uptake correlated inversely with the formation of tight junctions. Intracellular ICG was entrapped in the membrane traffic system, resulting in its slow turnover and prolonged retention by tumor cells. Our results suggest that tumor-specific imaging by ICG involves non-specific delivery of the dye to tissues followed by preferential tumor cellular uptake and retention. The tumor cell-preference of ICG is driven by passive tumor cell-targeting, the inherent ability of ICG to bind to cell membranes, and the high endocytic activity of tumor cells in association with the disruption of their tight junctions. © 2016 UICC.

Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and Nonphagocytic Cells in a Size-Dependent Manner.

PubMed

Cheng, Xiaju; Tian, Xin; Wu, Anqing; Li, Jianxiang; Tian, Jian; Chong, Yu; Chai, Zhifang; Zhao, Yuliang; Chen, Chunying; Ge, Cuicui

2015-09-23

The interaction at nanobio is a critical issue in designing safe nanomaterials for biomedical applications. Recent studies have reported that it is nanoparticle-protein corona rather than bare nanoparticle that determines the nanoparticle-cell interactions, including endocytic pathway and biological responses. Here, we demonstrate the effects of protein corona on cellular uptake of different sized gold nanoparticles in different cell lines. The experimental results show that protein corona significantly decreases the internalization of Au NPs in a particle size- and cell type-dependent manner. Protein corona exhibits much more significant inhibition on the uptake of large-sized Au NPs by phagocytic cell than that of small-sized Au NPs by nonphagocytic cell. The endocytosis experiment indicates that different endocytic pathways might be responsible for the differential roles of protein corona in the interaction of different sized Au NPs with different cell lines. Our findings can provide useful information for rational design of nanomaterials in biomedical application.

Ceramic nanoparticles: Recompense, cellular uptake and toxicity concerns.

PubMed

Singh, Deependra; Singh, Satpal; Sahu, Jageshwari; Srivastava, Shikha; Singh, Manju Rawat

2016-01-01

Over the past few years, nanoparticles and their role in drug delivery have been the centre of attraction as new drug delivery systems. Various forms of nanosystems have been designed, such as nanoclays, scaffolds and nanotubes, having numerous applications in areas such as drug loading, target cell uptake, bioassay and imaging. The present study discusses various types of nanoparticles, with special emphasis on ceramic nanocarriers. Ceramic materials have high mechanical strength, good body response and low or non-existing biodegradability. In this article, the various aspects concerning ceramic nanoparticles, such as their advantages over other systems, their cellular uptake and toxicity concerns are discussed in detail.

Cellular uptake of modified oligonucleotides: fluorescence approach

NASA Astrophysics Data System (ADS)

Kočišová, Eva; Praus, Petr; Rosenberg, Ivan; Seksek, Olivier; Sureau, Franck; Štěpánek, Josef; Turpin, Pierre-Yves

2005-06-01

Cellular uptake and intracellular distribution of the synthetic antisense analogue of dT 15 oligonucleotide (homogenously containing 3'-O-P-CH 2-O-5' internucleotide linkages and labeled with tetramethylrhodamine dye) was studied on B16 melanoma cell line by fluorescence micro-imaging and time-resolved microspectrofluorimetry. By using amphotericin B 3-dimethylaminopropyl amide as an enhancer molecule for the uptake process, homogenous staining of the cells with rather distinct nucleoli staining was achieved after 4 h of incubation. Two spectral components of 2.7 and 1.3 ns lifetime, respectively, were resolved in the emission collected from the cell nucleus. The way of staining and the long-lived component differed from our previous experiments demonstrating complexity of the intracellular oligonucleotide distribution and in particular of the binding inside the nucleus.

The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles

PubMed Central

Cho, Eun Chul; Zhang, Qiang; Xia, Younan

2011-01-01

In vitro experiments typically measure the uptake of nanoparticles by exposing cells at the bottom of a culture plate to a suspension of nanoparticles, which is assumed to be well-dispersed. However, nanoparticles can sediment and this means the concentration of particles on the cell surface and those actually taken up by the cells may be higher than the initial bulk concentration. Here we use upright and inverted cell culture configurations to show that cellular uptake of gold nanoparticles depends on the sedimentation and diffusion velocities of the nanoparticles and is independent of size, shape, density, surface coating and initial concentration of the nanoparticles. Generally more nanoparticles are taken up in the upright configuration than the inverted one and nanoparticles that sediment faster showed greater differences in uptake between the two configurations. Our results suggest that cellular uptake of nanoparticles is sensitive to the way cells are positioned and sedimentation need to be considered when performing in vitro studies for large and heavy nanoparticles. PMID:21516092

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

PubMed

Rodriguez-Lorenzo, Laura; Fytianos, Kleanthis; Blank, Fabian; von Garnier, Christophe; Rothen-Rutishauser, Barbara; Petri-Fink, Alke

2014-04-09

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy.

PubMed

Hondow, Nicole; Brown, M Rowan; Starborg, Tobias; Monteith, Alexander G; Brydson, Rik; Summers, Huw D; Rees, Paul; Brown, Andy

2016-02-01

Semiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalized particles are still to be achieved. We have focussed on the characterization of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and transmission electron microscopy to measure the final nanoparticle dose internalized by the U-2 OS cells. We show that a combination of analytical transmission electron microscopy and serial block face scanning electron microscopy can provide a comprehensive description of the internalization of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalized, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data-driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

Cellular uptake and transport of zein nanoparticles: effects of sodium caseinate.

PubMed

Luo, Yangchao; Teng, Zi; Wang, Thomas T Y; Wang, Qin

2013-08-07

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS)-stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with different zein/CAS mass ratios. The prepared nanoparticles demonstrated good stabilities to maintain particle size (120-140 nm) in cell culture medium and HBSS buffer at 37 °C. The nanoparticles showed no cytotoxicity for Caco-2 cells for 72 h. CAS not only significantly enhanced cell uptake of zein nanoparticles in a concentration- and time-dependent manner but also remarkably improved epithelial transport through Caco-2 cell monolayer. The cell uptake of zein-CAS nanoparticles indicated an energy-dependent endocytosis process as evidenced by cell uptake under blocking conditions, that is, 4 °C, sodium azide, and colchicine. Fluorescent microscopy clearly showed the internalization of zein-CAS nanoparticles. This study may shed some light on the cellular evaluations of hydrophobic protein nanoparticles.

Mycorrhizal phosphate uptake pathway in maize: vital for growth and cob development on nutrient poor agricultural and greenhouse soils

PubMed Central

Willmann, Martin; Gerlach, Nina; Buer, Benjamin; Polatajko, Aleksandra; Nagy, Réka; Koebke, Eva; Jansa, Jan; Flisch, René; Bucher, Marcel

2013-01-01

Arbuscular mycorrhizal fungi (AMF) form a mutually beneficial symbiosis with plant roots providing predominantly phosphorus in the form of orthophosphate (Pi) in exchange for plant carbohydrates on low P soils. The goal of this work was to generate molecular-genetic evidence in support of a major impact of the mycorrhizal Pi uptake (MPU) pathway on the productivity of the major crop plant maize under field and controlled conditions. Here we show, that a loss-of-function mutation in the mycorrhiza-specific Pi transporter gene Pht1;6 correlates with a dramatic reduction of above-ground biomass and cob production in agro-ecosystems with low P soils. In parallel mutant pht1;6 plants exhibited an altered fingerprint of chemical elements in shoots dependent on soil P availability. In controlled environments mycorrhiza development was impaired in mutant plants when grown alone. The presence of neighboring mycorrhizal nurse plants enhanced the reduced mycorrhiza formation in pht1;6 roots. Uptake of 33P-labeled orthophosphate via the MPU pathway was strongly impaired in colonized mutant plants. Moreover, repression of the MPU pathway resulted in a redirection of Pi to neighboring plants. In line with previous results, our data highlight the relevance of the MPU pathway in Pi allocation within plant communities and in particular the role of Pht1;6 for the establishment of symbiotic Pi uptake and for maize productivity and nutritional value in low-input agricultural systems. In a first attempt to identify cellular pathways which are affected by Pht1;6 activity, gene expression profiling via RNA-Seq was performed and revealed a set of maize genes involved in cellular signaling which exhibited differential regulation in mycorrhizal pht1;6 and control plants. The RNA data provided support for the hypothesis that fungal supply of Pi and/or Pi transport across Pht1;6 affects cell wall biosynthesis and hormone metabolism in colonized root cells. PMID:24409191

Point-particle method to compute diffusion-limited cellular uptake.

PubMed

Sozza, A; Piazza, F; Cencini, M; De Lillo, F; Boffetta, G

2018-02-01

We present an efficient point-particle approach to simulate reaction-diffusion processes of spherical absorbing particles in the diffusion-limited regime, as simple models of cellular uptake. The exact solution for a single absorber is used to calibrate the method, linking the numerical parameters to the physical particle radius and uptake rate. We study the configurations of multiple absorbers of increasing complexity to examine the performance of the method by comparing our simulations with available exact analytical or numerical results. We demonstrate the potential of the method to resolve the complex diffusive interactions, here quantified by the Sherwood number, measuring the uptake rate in terms of that of isolated absorbers. We implement the method in a pseudospectral solver that can be generalized to include fluid motion and fluid-particle interactions. As a test case of the presence of a flow, we consider the uptake rate by a particle in a linear shear flow. Overall, our method represents a powerful and flexible computational tool that can be employed to investigate many complex situations in biology, chemistry, and related sciences.

Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

PubMed

Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

2015-05-01

Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

Mapping cellular Fe-S cluster uptake and exchange reactions - divergent pathways for iron-sulfur cluster delivery to human ferredoxins.

PubMed

Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

2016-12-07

Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

Enhanced cellular uptake of size-separated lipophilic silicon nanoparticles

NASA Astrophysics Data System (ADS)

Kusi-Appiah, Aubrey E.; Mastronardi, Melanie L.; Qian, Chenxi; Chen, Kenneth K.; Ghazanfari, Lida; Prommapan, Plengchart; Kübel, Christian; Ozin, Geoffrey A.; Lenhert, Steven

2017-03-01

Specific size, shape and surface chemistry influence the biological activity of nanoparticles. In the case of lipophilic nanoparticles, which are widely used in consumer products, there is evidence that particle size and formulation influences skin permeability and that lipophilic particles smaller than 6 nm can embed in lipid bilayers. Since most nanoparticle synthetic procedures result in mixtures of different particles, post-synthetic purification promises to provide insights into nanostructure-function relationships. Here we used size-selective precipitation to separate lipophilic allyl-benzyl-capped silicon nanoparticles into monodisperse fractions within the range of 1 nm to 5 nm. We measured liposomal encapsulation and cellular uptake of the monodisperse particles and found them to have generally low cytotoxicities in Hela cells. However, specific fractions showed reproducibly higher cytotoxicity than other fractions as well as the unseparated ensemble. Measurements indicate that the cytotoxicity mechanism involves oxidative stress and the differential cytotoxicity is due to enhanced cellular uptake by specific fractions. The results indicate that specific particles, with enhanced suitability for incorporation into lipophilic regions of liposomes and subsequent in vitro delivery to cells, are enriched in certain fractions.

[Cellular uptake of TPS-L-carnitine synthesised as transporter-based renal targeting prodrug].

PubMed

Li, Li; Zhu, Di; Sun, Xun

2012-11-01

To synthesize transporter-based renal targeting prodrug TPS-L-Carnitine and to determine its cellular uptake in vitro. Triptolide (TP) was conjugated with L-carnitine using succinate as the linker to form TPS-L-Carnitine, which could be specifically recognized by OCTN2, a cationic transporter with high affinity to L-Carnitine and is highly expressed on the apical membrane of renal proximal tubule cells. Cellular uptake assays of the prodrug and its parent drug were performed on HK-2 cells, a human proximal tubule cell line, in different temperature, concentration and in the presence of competitive inhibitors. TPS-L-Carnitine was taken up into HK-2 cells in a saturable and temperature- and concentration-dependent manner. The uptake process could be inhibited by the competitive inhibitors. The uptake of TPS-L-Carnitine was significantly higher than that of TP at 37 degrees C in the same drug concentration. TPS-L-Carnitine was taken through endocytosis mediated by transporter. TPS-L-Carnitine provides a good renal targeting property and lays the foundation for further studies in vivo.

Winding through the WNT pathway during cellular development and demise.

PubMed

Li, F; Chong, Z Z; Maiese, K

2006-01-01

In slightly over a period of twenty years, our comprehension of the cellular and molecular mechanisms that govern the Wnt signaling pathway continue to unfold. The Wnt proteins were initially implicated in viral carcinogenesis experiments associated with mammary tumors, but since this period investigations focusing on the Wnt pathways and their transmembrane receptors termed Frizzled have been advanced to demonstrate the critical nature of Wnt for the development of a variety of cell populations as well as the potential of the Wnt pathway to avert apoptotic injury. In particular, Wnt signaling plays a significant role in both the cardiovascular and nervous systems during embryonic cell patterning, proliferation, differentiation, and orientation. Furthermore, modulation of Wnt signaling under specific cellular influences can either promote or prevent the early and late stages of apoptotic cellular injury in neurons, endothelial cells, vascular smooth muscle cells, and cardiomyocytes. A number of downstream signal transduction pathways can mediate the biological response of the Wnt proteins that include Dishevelled, beta-catenin, intracellular calcium, protein kinase C, Akt, and glycogen synthase kinase-3beta. Interestingly, these cellular cascades of the Wnt-Frizzled pathways can participate in several neurodegenerative, vascular, and cardiac disorders and may be closely integrated with the function of trophic factors. Identification of the critical elements that modulate the Wnt-Frizzled signaling pathway should continue to unlock the potential of Wnt pathway for the development of new therapeutic options against neurodegenerative and vascular diseases.

Novel mucus-penetrating liposomes as a potential oral drug delivery system: preparation, in vitro characterization, and enhanced cellular uptake

PubMed Central

Li, Xiuying; Chen, Dan; Le, Chaoyi; Zhu, Chunliu; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2011-01-01

Background The aim of this study was to investigate the intestinal mucus-penetrating properties and intestinal cellular uptake of two types of liposomes modified by Pluronic F127 (PF127). Methods The two types of liposomes, ie, PF127-inlaid liposomes and PF127-adsorbed liposomes, were prepared by a thin-film hydration method followed by extrusion, in which coumarin 6 was loaded as a fluorescence marker. A modified Franz diffusion cell mounted with the intestinal mucus of rats was used to study the diffusion characteristics of the two types of PF127 liposomes. Cell uptake studies were conducted in Caco-2 cells and analyzed using confocal laser scanning microcopy as well as flow cytometry. Results The diffusion efficiency of the two types of PF127-modified liposomes through intestinal rat mucus was 5–7-fold higher than that of unmodified liposomes. Compared with unmodified liposomes, PF127-inlaid liposomes showed significantly higher cellular uptake of courmarin 6. PF127-adsorbed liposomes showed a lower cellular uptake. Moreover, and interestingly, the two types of PF127-modified liposomes showed different cellular uptake mechanisms in Caco-2 cells. Conclusion PF127-inlaid liposomes with improved intestinal mucus-penetrating ability and enhanced cellular uptake might be a potential carrier candidate for oral drug delivery. PMID:22163166

Cellular Uptake of Tile-Assembled DNA Nanotubes.

PubMed

Kocabey, Samet; Meinl, Hanna; MacPherson, Iain S; Cassinelli, Valentina; Manetto, Antonio; Rothenfusser, Simon; Liedl, Tim; Lichtenegger, Felix S

2014-12-30

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP -expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

PubMed

O'Clock, George D

2016-08-01

Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

PubMed Central

2015-01-01

Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. PMID:24978295

Cellular Origin of [18F]FDG-PET Imaging Signals During Ceftriaxone-Stimulated Glutamate Uptake: Astrocytes and Neurons.

PubMed

Dienel, Gerald A; Behar, Kevin L; Rothman, Douglas L

2017-12-01

Ceftriaxone stimulates astrocytic uptake of the excitatory neurotransmitter glutamate, and it is used to treat glutamatergic excitotoxicity that becomes manifest during many brain diseases. Ceftriaxone-stimulated glutamate transport was reported to drive signals underlying [ 18 F]fluorodeoxyglucose-positron emission tomographic ([ 18 F]FDG-PET) metabolic images of brain glucose utilization and interpreted as supportive of the notion of lactate shuttling from astrocytes to neurons. This study draws attention to critical roles of astrocytes in the energetics and imaging of brain activity, but the results are provocative because (1) the method does not have cellular resolution or provide information about downstream pathways of glucose metabolism, (2) neuronal and astrocytic [ 18 F]FDG uptake were not separately measured, and (3) strong evidence against lactate shuttling was not discussed. Evaluation of potential metabolic responses to ceftriaxone suggests lack of astrocytic specificity and significant contributions by pre- and postsynaptic neuronal compartments. Indeed, astrocytic glycolysis may not make a strong contribution to the [ 18 F]FDG-PET signal because partial or complete oxidation of one glutamate molecule on its uptake generates enough ATP to fuel uptake of 3 to 10 more glutamate molecules, diminishing reliance on glycolysis. The influence of ceftriaxone on energetics of glutamate-glutamine cycling must be determined in astrocytes and neurons to elucidate its roles in excitotoxicity treatment.

Elementary Mode Analysis: A Useful Metabolic Pathway Analysis Tool for Characterizing Cellular Metabolism

PubMed Central

Trinh, Cong T.; Wlaschin, Aaron; Srienc, Friedrich

2010-01-01

Elementary Mode Analysis is a useful Metabolic Pathway Analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering. PMID:19015845

Cellular uptake of titanium and vanadium from addition of salts or fretting corrosion in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maurer, A.M.; Merritt, K.; Brown, S.A.

1994-02-01

The use of titanium and titanium-6% aluminum-4% vanadium alloy for dental and orthopedic implants has increased in the last decade. The implants are presumed to be compatible because oseointegration, bony apposition, and cell attachment are known. However, the cellular association of titanium and vanadium have remained unknown. This study examined the uptake of salts or fretting corrosion products. Titanium was not observed to be toxic to the cells. Vanadium was toxic at levels greater than 10[mu]g/mL. The percentage of cellular association of titanium was shown to be about 10 times that of vanadium. The percentage of cellular association of eithermore » element was greater from fretting corrosion than from the addition of salts. The presence of vanadium did not affect the cellular uptake of titanium. The presence of titanium decreased the cell association of vanadium.« less

[Cell signaling pathways interaction in cellular proliferation: Potential target for therapeutic interventionism].

PubMed

Valdespino-Gómez, Víctor Manuel; Valdespino-Castillo, Patricia Margarita; Valdespino-Castillo, Víctor Edmundo

2015-01-01

Nowadays, cellular physiology is best understood by analysing their interacting molecular components. Proteins are the major components of the cells. Different proteins are organised in the form of functional clusters, pathways or networks. These molecules are ordered in clusters of receptor molecules of extracellular signals, transducers, sensors and biological response effectors. The identification of these intracellular signaling pathways in different cellular types has required a long journey of experimental work. More than 300 intracellular signaling pathways have been identified in human cells. They participate in cell homeostasis processes for structural and functional maintenance. Some of them participate simultaneously or in a nearly-consecutive progression to generate a cellular phenotypic change. In this review, an analysis is performed on the main intracellular signaling pathways that take part in the cellular proliferation process, and the potential use of some components of these pathways as target for therapeutic interventionism are also underlined. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

PubMed

Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

2015-01-21

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles.

PubMed

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G(1) cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-15-1-0419 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...COVERED 1 Sep 2016 - 31 Aug 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal...inappropriate cell growth, fluid secretion, and dysregulation of cellular energy metabolism. The enzyme AMPK regulates a number of cellular pathways, including

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway.

PubMed

Lakhan, Ram; Said, Hamid M

2017-04-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr 78 Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway.

Lipopolysaccharide inhibits colonic biotin uptake via interference with membrane expression of its transporter: a role for a casein kinase 2-mediated pathway

PubMed Central

Lakhan, Ram

2017-01-01

Biotin (vitamin B7), an essential micronutrient for normal cellular functions, is obtained from both dietary sources as well as gut microbiota. Absorption of biotin in both the small and large intestine is via a carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT). Although different physiological and molecular aspects of intestinal biotin uptake have been delineated, nothing is known about the effect of LPS on the process. We addressed this issue using in vitro (human colonic epithelial NCM460 cells) and in vivo (mice) models of LPS exposure. Treating NCM460 cells with LPS was found to lead to a significant inhibition in carrier-mediated biotin uptake. Similarly, administration of LPS to mice led to a significant inhibition in biotin uptake by native colonic tissue. Although no changes in total cellular SMVT protein and mRNA levels were observed, LPS caused a decrease in the fraction of SMVT expressed at the cell surface. A role for casein kinase 2 (CK2) (whose activity was also inhibited by LPS) in mediating the endotoxin effects on biotin uptake and on membrane expression of SMVT was suggested by findings that specific inhibitors of CK2, as well as mutating the putative CK2 phosphorylation site (Thr78Ala) in the SMVT protein, led to inhibition in biotin uptake and membrane expression of SMVT. This study shows for the first time that LPS inhibits colonic biotin uptake via decreasing membrane expression of its transporter and that these effects likely involve a CK2-mediated pathway. PMID:28052864

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Learning cellular sorting pathways using protein interactions and sequence motifs.

PubMed

Lin, Tien-Ho; Bar-Joseph, Ziv; Murphy, Robert F

2011-11-01

Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/.

Learning Cellular Sorting Pathways Using Protein Interactions and Sequence Motifs

PubMed Central

Lin, Tien-Ho; Bar-Joseph, Ziv

2011-01-01

Abstract Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/. PMID:21999284

Effect of molecular characteristics on cellular uptake, subcellular localization, and phototoxicity of Zn(II) N-alkylpyridylporphyrins.

PubMed

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D; Batinic-Haberle, Ines; Benov, Ludmil T

2013-12-20

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.

Effect of Molecular Characteristics on Cellular Uptake, Subcellular Localization, and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins*

PubMed Central

Ezzeddine, Rima; Al-Banaw, Anwar; Tovmasyan, Artak; Craik, James D.; Batinic-Haberle, Ines; Benov, Ludmil T.

2013-01-01

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs. PMID:24214973

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

Cellular transport of l-arginine determines renal medullary blood flow in control rats, but not in diabetic rats despite enhanced cellular uptake capacity.

PubMed

Persson, Patrik; Fasching, Angelica; Teerlink, Tom; Hansell, Peter; Palm, Fredrik

2017-02-01

Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of l-arginine is rate limiting for nitric oxide production and that plasma l-arginine concentration is decreased in diabetes. We therefore investigated whether regional renal blood flow regulation is affected by cellular l-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital, and the left kidney was exposed. Total, cortical, and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either l-homoarginine to inhibit cellular uptake of l-arginine or N ω -nitro- l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. l-Homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. l-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both l-homoarginine and l-NAME were more pronounced in the control groups compared with the diabetic groups. Isolated cortical tubular cells displayed similar l-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased l-arginine uptake capacity. Diabetics had reduced l-arginine concentrations in plasma and medullary tissue but increased l-arginine concentration in cortical tissue. In conclusion, the reduced l-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular l-arginine and the upregulated cortical tissue l-arginine may protect cortical hemodynamics in diabetes. Copyright © 2017 the American Physiological Society.

An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.

PubMed

Digiacomo, L; Cardarelli, F; Pozzi, D; Palchetti, S; Digman, M A; Gratton, E; Capriotti, A L; Mahmoudi, M; Caracciolo, G

2017-11-16

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles

PubMed Central

Yang, Fang; Tang, Quanming; Zhong, Xueyun; Bai, Yan; Chen, Tianfeng; Zhang, Yibo; Li, Yinghua; Zheng, Wenjie

2012-01-01

A simple and solution-phase method for functionalization of selenium nanoparticles (SeNPs) with Spirulina polysaccharides (SPS) has been developed in the present study. The cellular uptake and anticancer activity of SPS-SeNPs were also evaluated. Monodisperse and homogeneous spherical SPS-SeNPs with diameters ranging from 20 nm to 50 nm were achieved under optimized conditions, which were stable in the solution phase for at least 3 months. SPS surface decoration significantly enhanced the cellular uptake and cytotoxicity of SeNPs toward several human cancer cell lines. A375 human melanoma cells were found extremely susceptible to SPS-SeNPs with half maximal (50%) inhibitory concentration value of 7.94 μM. Investigation of the underlying mechanisms revealed that SPS-SeNPs inhibited cancer cell growth through induction of apoptosis, as evidenced by an increase in sub-G1 cell population, deoxyribonucleic acid fragmentation, chromatin condensation, and phosphatidylserine translocation. Results suggest that the strategy to use SPS as a surface decorator could be an effective way to enhance the cellular uptake and anticancer efficacy of nanomaterials. SPS-SeNPs may be a potential candidate for further evaluation as a chemopreventive and chemotherapeutic agent against human cancers. PMID:22359460

Hippo Signaling: Key Emerging Pathway in Cellular and Whole-Body Metabolism.

PubMed

Ardestani, Amin; Lupse, Blaz; Maedler, Kathrin

2018-05-05

The evolutionarily conserved Hippo pathway is a key regulator of organ size and tissue homeostasis. Its dysregulation is linked to multiple pathological disorders. In addition to regulating development and growth, recent studies show that Hippo pathway components such as MST1/2 and LATS1/2 kinases, as well as YAP/TAZ transcriptional coactivators, are regulated by metabolic pathways and that the Hippo pathway controls metabolic processes at the cellular and organismal levels in physiological and metabolic disease states such as obesity, type 2 diabetes (T2D), nonalcoholic fatty liver disease (NAFLD), cardiovascular disorders, and cancer. In this review we summarize the connection between key Hippo components and metabolism, and how this interplay regulates cellular metabolism and metabolic pathways. The emerging function of Hippo in the regulation of metabolic homeostasis under physiological and pathological conditions is highlighted. Copyright © 2018 Elsevier Ltd. All rights reserved.

Correlation of Emulsion Structure with Cellular Uptake Behavior of Encapsulated Bioactive Nutrients: Influence of Droplet Size and Interfacial Structure.

PubMed

Lu, Wei; Kelly, Alan L; Maguire, Pierce; Zhang, Hongzhou; Stanton, Catherine; Miao, Song

2016-11-16

In this study, an in vitro Caco-2 cell culture assay was employed to evaluate the correlation between emulsion structure and cellular uptake of encapsulated β-carotene. After 4 h of incubation, an emulsion stabilized with whey protein isolate showed the highest intracellular accumulation of β-carotene (1.06 μg), followed by that stabilized with sodium caseinate (0.60 μg) and Tween 80 (0.20 μg), which are 13-, 7.5-, and 2.5-fold higher than that of free β-carotene (0.08 μg), respectively. Emulsions with small droplet size (239 ± 5 nm) showed a higher cellular uptake of β-carotene (1.56 μg) than emulsiond with large droplet size (489 ± 9 nm) (0.93 μg) (p < 0.01). The results suggested that delivery in an emulsion significantly improved the cellular uptake of β-carotene and thus potentially its bioavailability; uptake was closely correlated with the interfacial composition and droplet size of emulsions. The findings support the potential for achieving optimal controlled and targeted delivery of bioactive nutrients by structuring emulsions.

Bioaccessibility and Cellular Uptake of β-Carotene Encapsulated in Model O/W Emulsions: Influence of Initial Droplet Size and Emulsifiers

PubMed Central

Kelly, Alan L.

2017-01-01

The effects of the initial emulsion structure (droplet size and emulsifier) on the properties of β-carotene-loaded emulsions and the bioavailability of β-carotene after passing through simulated gastrointestinal tract (GIT) digestion were investigated. Exposure to GIT significantly changed the droplet size, surface charge and composition of all emulsions, and these changes were dependent on their initial droplet size and the emulsifiers used. Whey protein isolate (WPI)-stabilized emulsion showed the highest β-carotene bioaccessibility, while sodium caseinate (SCN)-stabilized emulsion showed the highest cellular uptake of β-carotene. The bioavailability of emulsion-encapsulated β-carotene based on the results of bioaccessibility and cellular uptake showed the same order with the results of cellular uptake being SCN > TW80 > WPI. An inconsistency between the results of bioaccessibility and bioavailability was observed, indicating that the cellular uptake assay is necessary for a reliable evaluation of the bioavailability of emulsion-encapsulated compounds. The findings in this study contribute to a better understanding of the correlation between emulsion structure and the digestive fate of emulsion-encapsulated nutrients, which make it possible to achieve controlled or potential targeted delivery of nutrients by designing the structure of emulsion-based carriers. PMID:28930195

Differential polymer structure tunes mechanism of cellular uptake and transfection routes of poly(β-amino ester) polyplexes in human breast cancer cells.

PubMed

Kim, Jayoung; Sunshine, Joel C; Green, Jordan J

2014-01-15

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10-17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60-90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

Cellular uptake and intracellular trafficking of PEG-b-PLA polymeric micelles.

PubMed

Zhang, Zhen; Xiong, Xiaoqin; Wan, Jiangling; Xiao, Ling; Gan, Lu; Feng, Youmei; Xu, Huibi; Yang, Xiangliang

2012-10-01

Besides as an inert carrier for hydrophobic anticancer agents, polymeric micelles composed of di-block copolymer poly(ethylene glycol)-poly(lactic acid) (PEG-b-PLA) function as biological response modifiers including reversal of multidrug resistance in cancer. However, the uptake mechanisms and the subsequent intracellular trafficking remain to be elucidated. In this paper, we found that the uptake of PEG-b-PLA polymeric micelles incorporating nile red (M-NR) was significantly inhibited by both dynamin inhibitor dynasore and dynamin-2 dominant negative mutant (dynamin-2 K44A). Exogenously expressed caveolin-1 colocalized with M-NR and upregulated M-NR internalization in HepG2 cells expressing low level of endogenous caveolin-1, while caveolin-1 dominant negative mutant (caveolin-1 Y14F) significantly downregulated M-NR internalization in C6 cells expressing high level of endogenous caveolin-1. Exogenously expressed clathrin light chain A (clathrin LCa) did not mainly colocalize with the internalized M-NR and had no effect on M-NR uptake. These results suggested that dynamin- and caveolin-dependent but clathrin-independent endocytosis was involved in M-NR cellular uptake. We further found that M-NR colocalized with lysosome and microtubulin after internalization. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combinatorics of feedback in cellular uptake and metabolism of small molecules.

PubMed

Krishna, Sandeep; Semsey, Szabolcs; Sneppen, Kim

2007-12-26

We analyze the connection between structure and function for regulatory motifs associated with cellular uptake and usage of small molecules. Based on the boolean logic of the feedback we suggest four classes: the socialist, consumer, fashion, and collector motifs. We find that the socialist motif is good for homeostasis of a useful but potentially poisonous molecule, whereas the consumer motif is optimal for nutrition molecules. Accordingly, examples of these motifs are found in, respectively, the iron homeostasis system in various organisms and in the uptake of sugar molecules in bacteria. The remaining two motifs have no obvious analogs in small molecule regulation, but we illustrate their behavior using analogies to fashion and obesity. These extreme motifs could inspire construction of synthetic systems that exhibit bistable, history-dependent states, and homeostasis of flux (rather than concentration).

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

PubMed Central

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-01-01

Background and Aims The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Methods Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. Key Results In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. Conclusions These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

PubMed

Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

2015-12-01

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Cellular uptake mechanisms of functionalised multi-walled carbon nanotubes by 3D electron tomography imaging

NASA Astrophysics Data System (ADS)

Al-Jamal, Khuloud T.; Nerl, Hannah; Müller, Karin H.; Ali-Boucetta, Hanene; Li, Shouping; Haynes, Peter D.; Jinschek, Joerg R.; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas; Porter, Alexandra E.

2011-06-01

Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed that MWNT-NH3+ were internalised in both phagocytic and non-phagocytic cells by any one of three mechanisms: (a) individually via membrane wrapping; (b) individually by direct membrane translocation; and (c) in clusters within vesicular compartments. At early time points following intracellular translocation, we noticed accumulation of nanotube material within various intracellular compartments, while a long-term (14-day) study using primary human macrophages revealed that MWNT-NH3+ were able to escape vesicular (phagosome) entrapment by translocating directly into the cytoplasm.Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed

Coating barium titanate nanoparticles with polyethylenimine improves cellular uptake and allows for coupled imaging and gene delivery

PubMed Central

Dempsey, Christopher; Lee, Isac; Cowan, Katie; Suh, Junghae

2015-01-01

Barium titanate nanoparticles (BT NP) belong to a class of second harmonic generating (SHG) nanoprobes that have recently demonstrated promise in biological imaging. Unfortunately, BT NPs display low cellular uptake efficiencies, which may be a problem if cellular internalization is desired or required for a particular application. To overcome this issue, while concomitantly developing a particle platform that can also deliver nucleic acids into cells, we coated the BT NPs with the cationic polymer polyethylenimine (PEI) – one of the most effective nonviral gene delivery agents. Coating of BT with PEI yielded complexes with positive zeta potentials and resulted in an 8-fold increase in cellular uptake of the BT NPs. Importantly, we were able to achieve high levels of gene delivery with the BT-PEI/DNA complexes, supporting further efforts to generate BT platforms for coupled imaging and gene therapy. PMID:23973999

Cellular uptake and metabolism of curcuminoids in monocytes/macrophages: regulatory effects on lipid accumulation

USDA-ARS?s Scientific Manuscript database

We previously showed that curcumin (CUR) may increase lipid accumulation in cultured THP-1 monocytes/macrophages, but tetrahydrocurcumin (THC), an in vivo metabolite of CUR, had no such effect. In the present study, we have hypothesized that different cellular uptake and/or metabolism of CUR and THC...

Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm, Bombyx mori

PubMed Central

Dong, Xiao-Long; Chai, Chun-Li; Pan, Cai-Xia; Tang, Hui; Chen, Yan-Hong; Dai, Fang-Yin; Pan, Min-Hui; Lu, Cheng

2014-01-01

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein. PMID:24475153

Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

NASA Astrophysics Data System (ADS)

Shah, Dhiral Ashwin

Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway

NASA Astrophysics Data System (ADS)

Goldstein, Alona; Soroka, Yoram; Frušić-Zlotkin, Marina; Lewis, Aaron; Kohen, Ron

2016-06-01

Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.

Hydrodynamic size-dependent cellular uptake of aqueous QDs probed by fluorescence correlation spectroscopy.

PubMed

Dong, Chaoqing; Irudayaraj, Joseph

2012-10-11

Aqueous quantum dots (QDs) directly synthesized with various thiol ligands have been investigated as imaging probes in living cells. However, the effect of the surface chemistry of these ligands on QDs' cellular uptakes and their intracellular fate remains poorly understood. In this work, four CdTe QDs were directly synthesized under aqueous conditions using four different thiols as stabilizers and their interactions with cells were investigated. Fluorescence correlation spectroscopy (FCS), X-ray photoelectron spectroscopy (XPS), and zeta potential measurements on QDs primarily show that the surface structure of these QDs is highly dependent on the thiol ligands used in the preparation of QDs' precursors, including its layer thicknesses, densities, and surface charges. Subsequently, FCS integrated with the maximum-entropy-method-based FCS (MEMFCS) was used to investigate the concentration distribution and dynamics of these QDs in living A-427 cells. Our findings indicate that QDs' surface characteristics affect cell membrane adsorption and subsequent internalization. More critically, we show that the cellular uptake of aqueous QDs is dependent on their hydrodynamic diameter and might have the potential to escape trapped environments to accumulate in the cytoplasm.

The role of endocytic pathways in cellular uptake of plasma non-transferrin iron

PubMed Central

Sohn, Yang-Sung; Ghoti, Hussam; Breuer, William; Rachmilewitz, Eliezer; Attar, Samah; Weiss, Guenter; Cabantchik, Z. Ioav

2012-01-01

Background In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis. Design and Methods Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes. Results Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r2=0.61) and also preventable by pre-chelation. Conclusions Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

2008-06-06

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. Wemore » propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.« less

Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

PubMed

Schrand, Amanda M; Lin, Jonathan B; Hens, Suzanne Ciftan; Hussain, Saber M

2011-02-01

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

Divergent cellular pathways of hippocampal memory consolidation and reconsolidation

PubMed Central

Lee, Jonathan L. C.; Hynds, Robert E.

2013-01-01

The reconsolidation of memories after their retrieval involves cellular mechanisms that recapitulate much of the initial consolidation process. However, we have previously demonstrated that there are independent cellular mechanisms of consolidation and reconsolidation in the dorsal hippocampus for contextual fear memories. Expression of BDNF was required for consolidation, while Zif268 expression was necessary for reconsolidation. Given the dichotomy between the obvious mechanistic similarity and notable dissociations between consolidation and reconsolidation, we sought to determine whether the separation at the level of gene expression reflected either parallel and independent upstream signalling pathways, or common upstream mechanisms that diverge by the level of transcriptional activation. Here we show that while consolidation and reconsolidation are commonly dependent upon NMDA receptor activation in the dorsal hippocampus there is a double dissociation between the effects of the MEK inhibitor U0126 and the IKK inhibitor sulfasalazine. Moreover, rescue experiments and western blot analyses show that there are functional NMDA receptor–ERK1–BDNF and NMDA receptor–IKKα–Zif268 pathways for consolidation and reconsolidation, respectively. Therefore, there are divergent pathways of hippocampal memory consolidation and reconsolidation, involving commonality at the cell surface, but separable downstream kinase cascades and transcriptional regulation. PMID:23197404

Construction and cellular uptake behavior of redox-sensitive docetaxel prodrug-loaded liposomes.

PubMed

Ren, Guolian; Jiang, Mengjuan; Guo, Weiling; Sun, Bingjun; Lian, He; Wang, Yongjun; He, Zhonggui

2018-01-01

A redox-responsive docetaxel (DTX) prodrug consisting of a disulfide linkage between DTX and vitamin E (DTX-SS-VE) was synthesized in our laboratory and was successfully formulated into liposomes. The aim of this study was to optimize the formulation and investigate the cellular uptake of DTX prodrug-loaded liposomes (DPLs). The content of DTX-SS-VE was determined by ultrahigh-performance liquid chromatography (UPLC). The formulation and process were optimized using entrapment efficiency (EE), drug-loading (DL), particle size and polydispersity index (PDI) as the evaluation indices. The optimal formulation was as follows: drug/lipid ratio of 1:12, cholesterol/lipid ratio of 1:10, hydration temperature of 40 °C, sonication power and time of 400 W and 5 min. The EE, DL and particle size of the optimized DPLs were 97.60 ± 0.03%, 7.09 ± 0.22% and 93.06 ± 0.72 nm, respectively. DPLs had good dilution stability under the physiological conditions over 24 h. In addition, DPLs were found to enter tumor cells via different pathways and released DTX from the prodrug to induce apoptosis. Taken together, the optimized formulation and process were found to be a simple, stable and applicable method for the preparation of DPLs that could successfully escape from lysosomes.

Cellular uptake and in vitro antitumor efficacy of composite liposomes for neutron capture therapy.

PubMed

Peters, Tanja; Grunewald, Catrin; Blaickner, Matthias; Ziegner, Markus; Schütz, Christian; Iffland, Dorothee; Hampel, Gabriele; Nawroth, Thomas; Langguth, Peter

2015-02-22

Neutron capture therapy for glioblastoma has focused mainly on the use of (10)B as neutron capture isotope. However, (157)Gd offers several advantages over boron, such as higher cross section for thermal neutrons and the possibility to perform magnetic resonance imaging during neutron irradiation, thereby combining therapy and diagnostics. We have developed different liposomal formulations of gadolinium-DTPA (Magnevist®) for application in neutron capture therapy of glioblastoma. The formulations were characterized physicochemically and tested in vitro in a glioma cell model for their effectiveness. Liposomes entrapping gadolinium-DTPA as neutron capture agent were manufactured via lipid/film-extrusion method and characterized with regard to size, entrapment efficiency and in vitro release. For neutron irradiation, F98 and LN229 glioma cells were incubated with the newly developed liposomes and subsequently irradiated at the thermal column of the TRIGA reactor in Mainz. The dose rate derived from neutron irradiation with (157)Gd as neutron capturing agent was calculated via Monte Carlo simulations and set in relation to the respective cell survival. The liposomal Gd-DTPA reduced cell survival of F98 and LN229 cells significantly. Differences in liposomal composition of the formulations led to distinctly different outcome in cell survival. The amount of cellular Gd was not at all times proportional to cell survival, indicating that intracellular deposition of formulated Gd has a major influence on cell survival. The majority of the dose contribution arises from photon cross irradiation compared to a very small Gd-related dose. Liposomal gadolinium formulations represent a promising approach for neutron capture therapy of glioblastoma cells. The liposome composition determines the uptake and the survival of cells following radiation, presumably due to different uptake pathways of liposomes and intracellular deposition of gadolinium-DTPA. Due to the small range of

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

PubMed

Brochez, V; Van Heuverswyn, D; Diniz, J A; De Potter, C R; Van den Eeckhout, E G

1999-05-01

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.

PubMed

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-09-01

The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS

Imaging of size-dependent uptake and identification of novel pathways in mouse Peyer's patches using fluorescent organosilica particles.

PubMed

Awaad, Aziz; Nakamura, Michihiro; Ishimura, Kazunori

2012-07-01

We investigated size-dependent uptake of fluorescent thiol-organosilica particles by Peyer's patches (PPs). We performed an oral single-particle administration (95, 130, 200, 340, 695 and 1050 nm) and a simultaneous dual-particle administration using 2 kinds of particles. Histological imaging and quantitative analysis revealed that particles taken up by the PP subepithelial dome were size dependent, and there was an optimal size range for higher uptake. Quantitative analysis of simultaneous dual-particle administration revealed that the percentage of fluorescence areas for 95, 130, 200, 340, 695 and 1050 nm with respect to 110 nm area was 124.0, 89.1, 73.8, 20.2, 9.2 and 0.5%, respectively. Additionally, imaging using fluorescent thiol-organosilica particles could detect 2 novel pathways through mouse PP epithelium: the transcellular pathway and the paracellular pathway. The uptake of nanoparticles based on an optimal size range and 2 novel pathways could indicate a new approach for vaccine delivery and nanomedicine development. Studying various sizes of fluorescent organosilica particles and their uptake in Peyer's patches, this team of authors determined the optimal size range of administration. Two novel pathways through mouse Peyer's patch epithelium were detected, i.e., the transcellular pathway and the paracellular pathway. This observation may have important applications in future vaccine delivery and nano-drug delivery. Copyright © 2012 Elsevier Inc. All rights reserved.

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.

PubMed

Li, Xiao C; Hopfer, Ulrich; Zhuo, Jia L

2009-11-01

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin

In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals.

PubMed

Holland, Jason P; Giansiracusa, Jeffrey H; Bell, Stephen G; Wong, Luet-Lok; Dilworth, Jonathan R

2009-04-07

The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [(60/62/64)Cu(II)ATSM] and [(60/62/64)Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO(2)-dependent in vitro cellular uptake and retention of [(64)Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k(1) = 9.8 +/- 0.59 x 10(-4) s(-1) and k(2) = 2.9 +/- 0.17 x 10(-3) s(-1)), intracellular reduction (k(3) = 5.2 +/- 0.31 x 10(-2) s(-1)), reoxidation (k(4) = 2.2 +/- 0.13 mol(-1) dm(3) s(-1)) and proton-mediated ligand dissociation (k(5) = 9.0 +/- 0.54 x 10(-5) s(-1)). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the

In vitro kinetic studies on the mechanism of oxygen-dependent cellular uptake of copper radiopharmaceuticals

NASA Astrophysics Data System (ADS)

Holland, Jason P.; Giansiracusa, Jeffrey H.; Bell, Stephen G.; Wong, Luet-Lok; Dilworth, Jonathan R.

2009-04-01

The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [60/62/64Cu(II)ATSM] and [60/62/64Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO2-dependent in vitro cellular uptake and retention of [64Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k1 = 9.8 ± 0.59 × 10-4 s-1 and k2 = 2.9 ± 0.17 × 10-3 s-1), intracellular reduction (k3 = 5.2 ± 0.31 × 10-2 s-1), reoxidation (k4 = 2.2 ± 0.13 mol-1 dm3 s-1) and proton-mediated ligand dissociation (k5 = 9.0 ± 0.54 × 10-5 s-1). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm

Cellular uptake of poly(allylamine hydrochloride) microcapsules with different deformability and its influence on cell functions.

PubMed

Yu, Wei; Zhang, Wenbo; Chen, Ying; Song, Xiaoxue; Tong, Weijun; Mao, Zhengwei; Gao, Changyou

2016-03-01

It is important to understand the safety issue and cell interaction pattern of polyelectrolyte microcapsules with different deformability before their use in biomedical applications. In this study, SiO2, poly(sodium-p-styrenesulfonate) (PSS) doped CaCO3 and porous CaCO3 spheres, all about 4μm in diameter, were used as templates to prepare microcapsules with different inner structure and subsequent deformability. As a result, three kinds of covalently assembled poly(allylaminehydrochloride)/glutaraldehyde (PAH/GA) microcapsules with similar size but different deformability under external osmotic pressure were prepared. The impact of different microcapsules on cell viability and functions are studied using smooth muscle cells (SMCs), endothelial cells (ECs) and HepG2 cells. The results demonstrated that viabilities of SMCs, ECs and HepG2 cells were not significantly influenced by either of the three kinds of microcapsules. However, the adhesion ability of SMCs and ECs as well as the mobility of SMCs, ECs and HepG2 cells were significantly impaired after treatment with microcapsules in a deformability dependent manner, especially the microcapsules with lower deformability caused higher impairment on cell functions. The cellular uptake kinetics, uptake pathways, intracellular distribution of microcapsules are further investigated in SMCs to reveal the potential mechanism. The SMCs showed faster uptake rate and exocytosis rate of microcapsules with lower deformability (Cap@CaCO3/PSS and Cap@CaCO3), leading to higher intracellular accumulation of microcapsules with lower deformability and possibly larger retardation of cell functions. The results pointed out that the deformability of microcapsules is an important factor governing the biological performance of microcapsules, which requires careful adjustment for further biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Deformable Hollow Periodic Mesoporous Organosilica Nanocapsules for Significantly Improved Cellular Uptake.

PubMed

Teng, Zhaogang; Wang, Chunyan; Tang, Yuxia; Li, Wei; Bao, Lei; Zhang, Xuehua; Su, Xiaodan; Zhang, Fan; Zhang, Junjie; Wang, Shouju; Zhao, Dongyuan; Lu, Guangming

2018-01-31

Mesoporous solids have been widely used in various biomedical areas such as drug delivery and tumor therapy. Although deformability has been recognized as a prime important characteristic influencing cellular uptake, the synthesis of deformable mesoporous solids is still a great challenge. Herein, deformable thioether-, benzene-, and ethane-bridged hollow periodic mesoporous organosilica (HPMO) nanocapsules have successfully been synthesized for the first time by a preferential etching approach. The prepared HPMO nanocapsules possess uniform diameters (240-310 nm), high surface areas (up to 878 m 2 ·g -1 ), well-defined mesopores (2.6-3.2 nm), and large pore volumes (0.33-0.75 m 3 ·g -1 ). Most importantly, the HPMO nanocapsules simultaneously have large hollow cavities (164-270 nm), thin shell thicknesses (20-38 nm), and abundant organic moiety in the shells, which endow a lower Young's modulus (E Y ) of 3.95 MPa than that of solid PMO nanoparticles (251 MPa). The HPMOs with low E Y are intrinsically flexible and deformable in the solution, which has been well-characterized by liquid cell electron microscopy. More interestingly, it is found that the deformable HPMOs can easily enter into human breast cancer MCF-7 cells via a spherical-to-oval morphology change, resulting in a 26-fold enhancement in cellular uptake (43.1% cells internalized with nanocapsules versus 1.65% cells with solid counterparts). The deformable HPMO nanocapsules were further loaded with anticancer drug doxorubicin (DOX), which shows high killing effects for MCF-7 cells, demonstrating the promise for biomedical applications.

Cellular and Molecular Pathways Leading to External Root Resorption

PubMed Central

Iglesias-Linares, A.; Hartsfield, J.K.

2016-01-01

External apical root resorption during orthodontic treatment implicates specific molecular pathways that orchestrate nonphysiologic cellular activation. To date, a substantial number of in vitro and in vivo molecular, genomic, and proteomic studies have supplied data that provide new insights into root resorption. Recent mechanisms and developments reviewed here include the role of the cellular component—specifically, the balance of CD68+, iNOS+ M1- and CD68+, CD163+ M2-like macrophages associated with root resorption and root surface repair processes linked to the expression of the M1-associated proinflammatory cytokine tumor necrosis factor, inducible nitric oxide synthase, the M1 activator interferon γ, the M2 activator interleukin 4, and M2-associated anti-inflammatory interleukin 10 and arginase I. Insights into the role of mesenchymal dental pulp cells in attenuating dentin resorption in homeostasis are also reviewed. Data on recently deciphered molecular pathways are reviewed at the level of (1) clastic cell adhesion in the external apical root resorption process and the specific role of α/β integrins, osteopontin, and related extracellular matrix proteins; (2) clastic cell fusion and activation by the RANKL/RANK/OPG and ATP-P2RX7-IL1 pathways; and (3) regulatory mechanisms of root resorption repair by cementum at the proteomic and transcriptomic levels. PMID:27811065

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Colpo, Pascal; Valsesia, Andrea; Urbán, Patricia; Ojea-Jiménez, Isaac; Gioria, Sabrina; Gilliland, Douglas; Rossi, François; Kinsner-Ovaskainen, Agnieszka

2015-01-01

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

PubMed Central

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Colpo, Pascal; Valsesia, Andrea; Urbán, Patricia; Ojea-Jiménez, Isaac; Gioria, Sabrina; Gilliland, Douglas; Rossi, François; Kinsner-Ovaskainen, Agnieszka

2015-01-01

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays. PMID:26517371

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

PubMed

Leclerc, L; Rima, W; Boudard, D; Pourchez, J; Forest, V; Bin, V; Mowat, P; Perriat, P; Tillement, O; Grosseau, P; Bernache-Assollant, D; Cottier, M

2012-08-01

Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Dysfunction of different cellular degradation pathways contributes to specific β-amyloid42-induced pathologies.

PubMed

Ji, Xuan-Ru; Cheng, Kuan-Chung; Chen, Yu-Ru; Lin, Tzu-Yu; Cheung, Chun Hei Antonio; Wu, Chia-Lin; Chiang, Hsueh-Cheng

2018-03-01

The endosomal-lysosomal system (ELS), autophagy, and ubiquitin-proteasome system (UPS) are cellular degradation pathways that each play a critical role in the removal of misfolded proteins and the prevention of the accumulation of abnormal proteins. Recent studies on Alzheimer's disease (AD) pathogenesis have suggested that accumulation of aggregated β-amyloid (Aβ) peptides in the AD brain results from a dysfunction in these cellular clearance systems. However, the specific roles of these pathways in the removal of Aβ peptides and the pathogenesis underlying AD are unclear. Our in vitro and in vivo genetic approaches revealed that ELS mainly removed monomeric β-amyloid42 (Aβ42), while autophagy and UPS clear oligomeric Aβ42. Although overproduction of phosphatidylinositol 4-phosphate-5 increased Aβ42 clearance, it reduced the life span of Aβ42 transgenic flies. Our behavioral studies further demonstrated impaired autophagy and UPS-enhanced Aβ42-induced learning and memory deficits, but there was no effect on Aβ42-induced reduction in life span. Results from genetic fluorescence imaging showed that these pathways were damaged in the following order: UPS, autophagy, and finally ELS. The results of our study demonstrate that different degradation pathways play distinct roles in the removal of Aβ42 aggregates and in disease progression. These findings also suggest that pharmacologic treatments that are designed to stimulate cellular degradation pathways in patients with AD should be used with caution.-Ji, X.-R., Cheng, K.-C., Chen, Y.-R., Lin, T.-Y., Cheung, C. H. A., Wu, C.-L., Chiang, H.-C. Dysfunction of different cellular degradation pathways contributes to specific β-amyloid42-induced pathologies.

Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability.

PubMed

Hu, Yun; Hoerle, Reece; Ehrich, Marion; Zhang, Chenming

2015-12-01

Lipid-polymer hybrid nanoparticles (NPs), consisting of a polymeric core and a lipid shell, have been intensively examined as delivery systems for cancer drugs, imaging agents, and vaccines. For applications in vaccine particularly, the hybrid NPs need to be able to protect the enclosed antigens during circulation, easily be up-taken by dendritic cells, and possess good stability for prolonged storage. However, the influence of lipid composition on the performance of hybrid NPs has not been well studied. In this study, we demonstrate that higher concentrations of cholesterol in the lipid layer enable slower and more controlled antigen release from lipid-poly(lactide-co-glycolide) acid (lipid-PLGA) NPs in human serum and phosphate buffered saline (PBS). Higher concentrations of cholesterol also promoted in vitro cellular uptake of hybrid NPs, improved the stability of the lipid layer, and protected the integrity of the hybrid structure during long-term storage. However, stabilized hybrid structures of high cholesterol content tended to fuse with each other during storage, resulting in significant size increase and lowered cellular uptake. Additional experiments demonstrated that PEGylation of NPs could effectively minimize fusion-caused size increase after long term storage, leading to improved cellular uptake, although excessive PEGylation will not be beneficial and led to reduced improvement. This paper reports the engineering of the lipid layer that encloses a polymeric nanoparticle, which can be used as a carrier for drug and vaccine molecules for targeted delivery. We demonstrated that the concentration of cholesterol is critical for the stability and uptake of the hybrid nanoparticles by dendritic cells, a targeted cell for the delivery of immune effector molecules. However, we found that hybrid nanoparticles with high cholesterol concentration tend to fuse during storage resulting in larger particles with decreased cellular uptake. This problem is

Membrane Microdomain Structures of Liposomes and Their Contribution to the Cellular Uptake Efficiency into HeLa Cells.

PubMed

Onuki, Yoshinori; Obata, Yasuko; Kawano, Kumi; Sano, Hiromu; Matsumoto, Reina; Hayashi, Yoshihiro; Takayama, Kozo

2016-02-01

The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters' liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains.

Cellular entry of G3.5 poly (amido amine) dendrimers by clathrin- and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia.

PubMed

Goldberg, Deborah S; Ghandehari, Hamidreza; Swaan, Peter W

2010-08-01

This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

PubMed Central

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-01-01

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers. PMID:27531648

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

NASA Astrophysics Data System (ADS)

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-08-01

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers.

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles.

PubMed

Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-08-17

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers.

Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs

PubMed Central

Xia, Tian; Kovochich, Michael; Liong, Monty; Meng, Huan; Kabehie, Sanaz; Zink, Jeffrey I.; Nel, Andre E.

2014-01-01

Surface-functionalized mesoporous silica nanoparticles (MSNP) can be used as an efficient and safe carrier for bioactive molecules. In order to make the MSNP a more efficient delivery system, we modified the surface of the particles by a functional group that enhances cellular uptake and allows nucleic acid delivery in addition to traditional drug delivery. Non-covalent attachment of polyethyleneimine (PEI) polymers to the surface not only increases MSNP cellular uptake, but also generates a cationic surface to which DNA and siRNA constructs could be attached. While efficient for intracellular delivery of these nucleic acids, the 25 KD PEI polymer unfortunately changes the safety profile of the MSNP that is otherwise very safe. By experimenting with several different polymer molecular weights, it was possible to retain high cellular uptake and transfection efficiency while reducing or even eliminating cationic MSNP cytotoxicity. The particles coated with the 10 KD PEI polymer was particularly efficient for transducing HEPA-1 cells with a siRNA construct that was capable of knocking down GFP expression. Similarly, transfection of a GFP plasmid induced effective expression of the fluorescent protein in > 70% cells in the population. These outcomes were quantitatively assessed by confocal microscopy and flow cytometry. We also demonstrated that the enhanced cellular uptake of the non-toxic cationic MSNP enhance the delivery of the hydrophobic anticancer drug, paclitaxel, to pancreatic cancer cells. In summary, we demonstrate that by a careful selection of PEI size, it is possible to construct cationic MSNP that are capable of nucleotide and enhanced drug delivery with minimal or no cytotoxicity. This novel use of a cationic MSNP extends its therapeutic use potential. PMID:19739605

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment.

PubMed

Ye, Guihua; Jiang, Yajun; Yang, Xiaoying; Hu, Hongxiang; Wang, Beibei; Sun, Lu; Yang, Victor C; Sun, Duxin; Gao, Wei

2018-01-10

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pH e : 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pH endo : 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux.

PubMed

Ost, Mario; Keipert, Susanne; van Schothorst, Evert M; Donner, Verena; van der Stelt, Inge; Kipp, Anna P; Petzke, Klaus-Jürgen; Jove, Mariona; Pamplona, Reinald; Portero-Otin, Manuel; Keijer, Jaap; Klaus, Susanne

2015-04-01

Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle. © FASEB.

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Enhancement of cellular uptake and cytotoxicity of curcumin-loaded PLGA nanoparticles by conjugation with anti-P-glycoprotein in drug resistance cancer cells.

PubMed

Punfa, Wanisa; Yodkeeree, Supachai; Pitchakarn, Pornsiri; Ampasavate, Chadarat; Limtrakul, Pornngarm

2012-06-01

To compare the anti-cancer activity and cellular uptake of curcumin (Cur) delivered by targeted and non-targeted drug delivery systems in multidrug-resistant cervical cancer cells. Cur was entrapped into poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Cur-NPs) in the presence of modified-pluronic F127 stabilizer using nano-precipitation technique. On the surface of Cur-NPs, the carboxy-terminal of modified pluronic F127 was conjugated to the amino-terminal of anti-P-glycoprotein (P-gp) (Cur-NPs-APgp). The physical properties of the Cur-NPs, including particle size, zeta potential, particle morphology and Cur release kinetics, were investigated. Cellular uptake and specificity of the Cur-NPs and Cur-NPs-APgp were detected in cervical cancer cell lines KB-V1 (higher expression of P-gp) and KB-3-1 (lower expression of P-gp) using fluorescence microscope and flow cytometry, respectively. Cytotoxicity of the Cur-NPs and Cur-NPs-APgp was determined using MTT assay. The particle size of Cur-NPs and Cur-NPs-APgp was 127 and 132 nm, respectively. The entrapment efficiency and actual loading of Cur-NPs-APgp (60% and 5 μg Cur/mg NP) were lower than those of Cur-NPs (99% and 7 μg Cur/mg NP). The specific binding of Cur-NPs-APgp to KB-V1 cells was significantly higher than that to KB-3-1 cells. Cellular uptake of Cur-NPs-APgp into KB-V1 cells was higher, as compared to KB-3-1 cells. However, the cellular uptake of Cur-NPs and Cur-NPs-IgG did not differ between the two types of cells. Besides, the cytotoxicity of Cur-NPs-APgp in KB-V1 cells was higher than those of Cur and Cur-NPs. The results demonstrate that Cur-NPs-APgp targeted to P-gp on the cell surface membrane of KB-V1 cells, thus enhancing the cellular uptake and cytotoxicity of Cur.

Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hag Dong; Jang, Chang-Young; Choe, Jeong Min

2012-06-01

Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that canmore » reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.« less

Surface chemistry of gold nanoparticles determines the biocorona composition impacting cellular uptake, toxicity and gene expression profiles in human endothelial cells.

PubMed

Chandran, Parwathy; Riviere, Jim E; Monteiro-Riviere, Nancy A

2017-05-01

This study investigated the role of nanoparticle size and surface chemistry on biocorona composition and its effect on uptake, toxicity and cellular responses in human umbilical vein endothelial cells (HUVEC), employing 40 and 80 nm gold nanoparticles (AuNP) with branched polyethyleneimine (BPEI), lipoic acid (LA) and polyethylene glycol (PEG) coatings. Proteomic analysis identified 59 hard corona proteins among the various AuNP, revealing largely surface chemistry-dependent signature adsorbomes exhibiting human serum albumin (HSA) abundance. Size distribution analysis revealed the relative instability and aggregation inducing potential of bare and corona-bound BPEI-AuNP, over LA- and PEG-AuNP. Circular dichroism analysis showed surface chemistry-dependent conformational changes of proteins binding to AuNP. Time-dependent uptake of bare, plasma corona (PC) and HSA corona-bound AuNP (HSA-AuNP) showed significant reduction in uptake with PC formation. Cell viability studies demonstrated dose-dependent toxicity of BPEI-AuNP. Transcriptional profiling studies revealed 126 genes, from 13 biological pathways, to be differentially regulated by 40 nm bare and PC-bound BPEI-AuNP (PC-BPEI-AuNP). Furthermore, PC formation relieved the toxicity of cationic BPEI-AuNP by modulating expression of genes involved in DNA damage and repair, heat shock response, mitochondrial energy metabolism, oxidative stress and antioxidant response, and ER stress and unfolded protein response cascades, which were aberrantly expressed in bare BPEI-AuNP-treated cells. NP surface chemistry is shown to play the dominant role over size in determining the biocorona composition, which in turn modulates cell uptake, and biological responses, consequently defining the potential safety and efficacy of nanoformulations.

Elucidating the Function of Penetratin and a Static Magnetic Field in Cellular Uptake of Magnetic Nanoparticles

PubMed Central

Chaudhary, Suman; Smith, Carol Anne; del Pino, Pablo; de la Fuente, Jesus M.; Mullin, Margaret; Hursthouse, Andrew; Stirling, David; Berry, Catherine C.

2013-01-01

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. PMID:24275948

Local and distal effects of arbuscular mycorrhizal colonization on direct pathway Pi uptake and root growth in Medicago truncatula

PubMed Central

Watts-Williams, Stephanie J.; Jakobsen, Iver; Cavagnaro, Timothy R.; Grønlund, Mette

2015-01-01

Two pathways exist for plant Pi uptake from soil: via root epidermal cells (direct pathway) or via associations with arbuscular mycorrhizal (AM) fungi, and the two pathways interact in a complex manner. This study investigated distal and local effects of AM colonization on direct root Pi uptake and root growth, at different soil P levels. Medicago truncatula was grown at three soil P levels in split-pots with or without AM fungal inoculation and where one root half grew into soil labelled with 33P. Plant genotypes included the A17 wild type and the mtpt4 mutant. The mtpt4 mutant, colonized by AM fungi, but with no functional mycorrhizal pathway for Pi uptake, was included to better understand effects of AM colonization per se. Colonization by AM fungi decreased expression of direct Pi transporter genes locally, but not distally in the wild type. In mtpt4 mutant plants, direct Pi transporter genes and the Pi starvation-induced gene Mt4 were more highly expressed than in wild-type roots. In wild-type plants, less Pi was taken up via the direct pathway by non-colonized roots when the other root half was colonized by AM fungi, compared with non-mycorrhizal plants. Colonization by AM fungi strongly influenced root growth locally and distally, and direct root Pi uptake activity locally, but had only a weak influence on distal direct pathway activity. The responses to AM colonization in the mtpt4 mutant suggested that in the wild type, the increased P concentration of colonized roots was a major factor driving the effects of AM colonization on direct root Pi uptake. PMID:25944927

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12

PubMed Central

Quadros, Edward V.; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Hannibal, Luciana; Wang, Sihe; Jacobsen, Donald W.; Fedosov, Sergey; Wright, Erica; Gallagher, Renata C.; Anastasio, Natascia; Watkins, David; Rosenblatt, David S.

2010-01-01

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function. PMID:20524213

Targeting cancer by binding iron: Dissecting cellular signaling pathways

PubMed Central

Lui, Goldie Y.L.; Kovacevic, Zaklina; Richardson, Vera; Merlot, Angelica M.; Kalinowski, Danuta S.; Richardson, Des R.

2015-01-01

Newer and more potent therapies are urgently needed to effectively treat advanced cancers that have developed resistance and metastasized. One such strategy is to target cancer cell iron metabolism, which is altered compared to normal cells and may facilitate their rapid proliferation. This is supported by studies reporting the anti-neoplastic activities of the clinically available iron chelators, desferrioxamine and deferasirox. More recently, ligands of the di-2-pyridylketone thiosemicarbazone (DpT) class have demonstrated potent and selective anti-proliferative activity across multiple cancer-types in vivo, fueling studies aimed at dissecting their molecular mechanisms of action. In the past five years alone, significant advances have been made in understanding how chelators not only modulate cellular iron metabolism, but also multiple signaling pathways implicated in tumor progression and metastasis. Herein, we discuss recent research on the targeting of iron in cancer cells, with a focus on the novel and potent DpT ligands. Several key studies have revealed that iron chelation can target the AKT, ERK, JNK, p38, STAT3, TGF-β, Wnt and autophagic pathways to subsequently inhibit cellular proliferation, the epithelial-mesenchymal transition (EMT) and metastasis. These developments emphasize that these novel therapies could be utilized clinically to effectively target cancer. PMID:26125440

Mechanisms of cellular uptake, intracellular transportation, and degradation of CIGB-300, a Tat-conjugated peptide, in tumor cell lines.

PubMed

Benavent Acero, Fernando R; Perera Negrin, Yasser; Alonso, Daniel F; Perea, Silvio E; Gomez, Daniel E; Farina, Hernán G

2014-06-02

CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate's phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

PubMed

Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

2017-03-10

Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

PubMed Central

Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

2017-01-01

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

Distribution and uptake pathways of organochlorine pesticides in greenhouse and conventional vegetables.

PubMed

Zhang, Anping; Luo, Wenxiu; Sun, Jianqiang; Xiao, Hang; Liu, Weiping

2015-02-01

The application of greenhouse vegetable cultivation has dramatically expanded worldwide during the last several decades. However, little information is available on the distribution and uptake of pesticides in greenhouse vegetables. To bridge this knowledge gap, the present study was initiated to investigate the distribution and uptake of organochlorine pesticides (OCPs) in vegetables from plastic greenhouse and conventional cultivation methods. The uptake pathways of OCPs were not significantly different between these two cultivation methods. The arithmetic means of OCP concentrations in greenhouse vegetables were higher than those in conventional vegetables, although there was no significant difference. This small difference raised the concern of whether the tiny difference could be magnified to a significant difference by bioaccumulation in the food chain. The issue should be addressed by a well-designed scheme in future studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

PubMed Central

Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

2015-01-01

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

PATIKA: an integrated visual environment for collaborative construction and analysis of cellular pathways.

PubMed

Demir, E; Babur, O; Dogrusoz, U; Gursoy, A; Nisanci, G; Cetin-Atalay, R; Ozturk, M

2002-07-01

Availability of the sequences of entire genomes shifts the scientific curiosity towards the identification of function of the genomes in large scale as in genome studies. In the near future, data produced about cellular processes at molecular level will accumulate with an accelerating rate as a result of proteomics studies. In this regard, it is essential to develop tools for storing, integrating, accessing, and analyzing this data effectively. We define an ontology for a comprehensive representation of cellular events. The ontology presented here enables integration of fragmented or incomplete pathway information and supports manipulation and incorporation of the stored data, as well as multiple levels of abstraction. Based on this ontology, we present the architecture of an integrated environment named Patika (Pathway Analysis Tool for Integration and Knowledge Acquisition). Patika is composed of a server-side, scalable, object-oriented database and client-side editors to provide an integrated, multi-user environment for visualizing and manipulating network of cellular events. This tool features automated pathway layout, functional computation support, advanced querying and a user-friendly graphical interface. We expect that Patika will be a valuable tool for rapid knowledge acquisition, microarray generated large-scale data interpretation, disease gene identification, and drug development. A prototype of Patika is available upon request from the authors.

Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

PubMed

Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

2016-09-22

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

Insights on how the Mycobacterium tuberculosis heme uptake pathway can be used as a drug target

PubMed Central

Owens, Cedric P; Chim, Nicholas; Goulding, Celia W

2013-01-01

Mycobacterium tuberculosis (Mtb) acquires non-heme iron through salicylate-derived siderophores termed mycobactins whereas heme iron is obtained through a cascade of heme uptake proteins. Three proteins are proposed to mediate Mtb heme iron uptake, a secreted heme transporter (Rv0203), and MmpL3 and MmpL11, which are potential transmembrane heme transfer proteins. Furthermore, MhuD, a cytoplasmic heme-degrading enzyme, has been identified. Rv0203, MmpL3 and MmpL11 are mycobacteria-specific proteins, making them excellent drug targets. Importantly, MmpL3, a necessary protein, has also been implicated in trehalose monomycolate export. Recent drug-discovery efforts revealed that MmpL3 is the target of several compounds with antimycobacterial activity. Inhibition of the Mtb heme uptake pathway has yet to be explored. We propose that inhibitor design could focus on heme analogs, with the goal of blocking specific steps of this pathway. In addition, heme uptake could be hijacked as a method of importing drugs into the mycobacterial cytosol. PMID:23919550

Cytotoxicity and cellular uptake of different sized gold nanoparticles in ovarian cancer cells

NASA Astrophysics Data System (ADS)

Kumar, Dhiraj; Mutreja, Isha; Chitcholtan, Kenny; Sykes, Peter

2017-11-01

Nanomedicine has advanced the biomedical field with the availability of multifunctional nanoparticles (NPs) systems that can target a disease site enabling drug delivery and helping to monitor the disease. In this paper, we synthesised the gold nanoparticles (AuNPs) with an average size 18, 40, 60 and 80 nm, and studied the effect of nanoparticles size, concentration and incubation time on ovarian cancer cells namely, OVCAR5, OVCAR8, and SKOV3. The size measured by transmission electron microscopy images was slightly smaller than the hydrodynamic diameter; measured size by ImageJ as 14.55, 38.13, 56.88 and 78.56 nm. The cellular uptake was significantly controlled by the AuNPs size, concentration, and the cell type. The nanoparticles uptake increased with increasing concentration, and 18 and 80 nm AuNPs showed higher uptake ranging from 1.3 to 5.4 μg depending upon the concentration and cell type. The AuNPs were associated with a temporary reduction in metabolic activity, but metabolic activity remained more than 60% for all sample types; NPs significantly affected the cell proliferation activity in first 12 h. The increase in nanoparticle size and concentration induced the production of reactive oxygen species in 24 h.

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death.

PubMed

Movafegh, Bahareh; Jalal, Razieh; Mohammadi, Zobeideh; Aldaghi, Seyyede Araste

2018-04-11

Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide-acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicin-induced cell death. Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24 h combined treatment of cells with doxorubicin (0.5 μM) and poly-L-arginine (1 μg ml-1) caused a small increase in doxorubicin-induced apoptosis and significant elevated necrosis in DU145 cells as compared to each agent alone. Conlusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferation-inducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

PubMed

Sorokin, Andrey

2016-01-01

The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential

Diffusion and cellular uptake of drugs in live cells studied with surface-enhanced Raman scattering probes

NASA Astrophysics Data System (ADS)

Bálint, Štefan; Rao, Satish; Sánchez, Mónica Marro; Huntošová, Veronika; Miškovský, Pavol; Petrov, Dmitri

2010-03-01

An understanding of the mechanisms of drug diffusion and uptake through cellular membranes is critical for elucidating drug action and in the development of effective drug delivery systems. We study these processes for emodin, a potential anticancer drug, in live cancer cells using surface-enhanced Raman scattering. Micrometer-sized silica beads covered by nanosized silver colloids are passively embedded into the cell and used as sensors of the drug. We demonstrate that the technique offers distinct advantages: the possibility to study the kinetics of drug diffusion through the cellular membrane toward specific cell organelles, the detection of lower drug concentrations compared to fluorescence techniques, and less damage imparted on the cell.

Cellular Metabolic and Autophagic Pathways: Traffic Control by Redox Signaling

PubMed Central

Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

2013-01-01

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality, and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function. PMID:23702245

Exploring cellular uptake of iron oxide nanoparticles associated with rhodium citrate in breast cancer cells.

PubMed

Chaves, Natalia L; Estrela-Lopis, Irina; Böttner, Julia; Lopes, Cláudio Ap; Guido, Bruna C; de Sousa, Aparecido R; Báo, Sônia N

2017-01-01

Nanocarriers have the potential to improve the therapeutic index of currently available drugs by improving their efficacy and achieving therapeutic steady-state levels over an extended period. The association of maghemite-rhodium citrate (MRC) nanoparticles (NPs) has the potential to increase specificity of the cytotoxic action. However, the interaction of these NPs with cells, their uptake mechanism, and subcellular localization need to be elucidated. This work evaluates the uptake mechanism of MRC NPs in metastatic and nonmetastatic breast cancer-cell models, comparing them to a nontumor cell line. MRC NPs uptake in breast cancer cells was more effective than in normal cells, with regard to both the amount of internalized material and the achievement of more strategic intracellular distribution. Moreover, this process occurred through a clathrin-dependent endocytosis pathway with different basal expression levels of this protein in the cell lines tested.

Bridging the gap between high-throughput genetic and transcriptional data reveals cellular pathways responding to alpha-synuclein toxicity

PubMed Central

Yeger-Lotem, Esti; Riva, Laura; Su, Linhui Julie; Gitler, Aaron D.; Cashikar, Anil; King, Oliver D.; Auluck, Pavan K.; Geddie, Melissa L.; Valastyan, Julie S.; Karger, David R.; Lindquist, Susan; Fraenkel, Ernest

2009-01-01

Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alpha-synuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways. PMID:19234470

Exploring the cellular and tissue uptake of nanomaterials in a range of biological samples using multimodal nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Johnston, Helinor J.; Mouras, Rabah; Brown, David M.; Elfick, Alistair; Stone, Vicki

2015-12-01

The uptake of nanomaterials (NMs) by cells is critical in determining their potential biological impact, whether beneficial or detrimental. Thus, investigation of NM internalization by cells is a common consideration in hazard and efficacy studies. There are currently a number of approaches that are routinely used to investigate NM-cell interactions, each of which have their own advantages and limitations. Ideally, imaging modalities used to investigate NM uptake by cells should not require the NM to be labelled (e.g. with fluorophores) to facilitate its detection. We present a multimodal imaging approach employing a combination of label-free microscopies that can be used to investigate NM-cell interactions. Coherent anti-Stokes Raman scattering microscopy was used in combination with either two-photon photoluminescence or four-wave mixing (FWM) to visualize the uptake of gold or titanium dioxide NMs respectively. Live and fixed cell imaging revealed that NMs were internalized by J774 macrophage and C3A hepatocyte cell lines (15-31 μg ml-1). Sprague Dawley rats were exposed to NMs (intratracheal instillation, 62 μg) and NMs were detected in blood and lung leucocytes, lung and liver tissue, demonstrating that NMs could translocate from the exposure site. Obtained data illustrate that multimodal nonlinear optical microscopy may help overcome current challenges in the assessment of NM cellular uptake and biodistribution. It is therefore a powerful tool that can be used to investigate unlabelled NM cellular and tissue uptake in three dimensions, requires minimal sample preparation, and is applicable to live and fixed cells.

Cellular Uptake and Mode-of-Action of Clostridium difficile Toxins.

PubMed

Papatheodorou, Panagiotis; Barth, Holger; Minton, Nigel; Aktories, Klaus

2018-01-01

Research on the human gut pathogen Clostridium difficile and its toxins has gained much attention, particularly as a consequence of the increasing threat to human health presented by emerging hypervirulent strains. Toxin A (TcdA) and B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (Clostridium difficile transferase). As C. difficile toxins are the causative agents of C. difficile-associated diseases (CDAD), such as antibiotics-associated diarrhea and pseudomembranous colitis, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Notably, a high proportion of studies on C. difficile toxins were performed in European laboratories. In this chapter we will highlight important recent advances in C. difficile toxins research.

Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake

NASA Astrophysics Data System (ADS)

Schöttler, S.; Klein, Katja; Landfester, K.; Mailänder, V.

2016-03-01

Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance

Uptake of leptin and albumin via separate pathways in proximal tubule cells.

PubMed

Briffa, Jessica F; Grinfeld, Esther; Poronnik, Philip; McAinch, Andrew J; Hryciw, Deanne H

2016-10-01

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5min exposure, however there was no co-localisation at 10, 20 and 30min exposure. In OK cells, acute exposure to leptin for 2h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Design strategy of pH-sensitive triblock copolymer micelles for efficient cellular uptake by computer simulations

NASA Astrophysics Data System (ADS)

Xia, Qiang-sheng; Ding, Hong-ming; Ma, Yu-qiang

2018-03-01

Efficient delivery of nanoparticles into specific cell interiors is of great importance in biomedicine. Recently, the pH-responsive micelle has emerged as one potential nanocarrier to realize such purpose since there exist obvious pH differences between normal tissues and tumors. Herein, by using dissipative particle dynamics simulation, we investigate the interaction of the pH-sensitive triblock copolymer micelles composed of ligand (L), hydrophobic block (C) and polyelectrolyte block (P) with cell membrane. It is found that the structure rearrangement of the micelle can facilitate its penetration into the lower leaflet of the bilayer. However, when the ligand-receptor specific interaction is weak, the micelles may just fuse with the upper leaflet of the bilayer. Moreover, the ionization degree of polyelectrolyte block and the length of hydrophobic block also play a vital role in the penetration efficiency. Further, when the sequence of the L, P, C beads in the copolymers is changed, the translocation pathways of the micelles may change from direct penetration to Janus engulfment. The present study reveals the relationship between the molecular structure of the copolymer and the uptake of the pH-sensitive micelles, which may give some significant insights into the experimental design of responsive micellar nanocarriers for highly efficient cellular delivery.

Exploring cellular uptake of iron oxide nanoparticles associated with rhodium citrate in breast cancer cells

PubMed Central

Chaves, Natalia L; Estrela-Lopis, Irina; Böttner, Julia; Lopes, Cláudio AP; Guido, Bruna C; de Sousa, Aparecido R; Báo, Sônia N

2017-01-01

Nanocarriers have the potential to improve the therapeutic index of currently available drugs by improving their efficacy and achieving therapeutic steady-state levels over an extended period. The association of maghemite–rhodium citrate (MRC) nanoparticles (NPs) has the potential to increase specificity of the cytotoxic action. However, the interaction of these NPs with cells, their uptake mechanism, and subcellular localization need to be elucidated. This work evaluates the uptake mechanism of MRC NPs in metastatic and nonmetastatic breast cancer-cell models, comparing them to a nontumor cell line. MRC NPs uptake in breast cancer cells was more effective than in normal cells, with regard to both the amount of internalized material and the achievement of more strategic intracellular distribution. Moreover, this process occurred through a clathrin-dependent endocytosis pathway with different basal expression levels of this protein in the cell lines tested. PMID:28814867

Comparison between Arabidopsis and Rice for Main Pathways of K(+) and Na(+) Uptake by Roots.

PubMed

Nieves-Cordones, Manuel; Martínez, Vicente; Benito, Begoña; Rubio, Francisco

2016-01-01

K(+) is an essential macronutrient for plants. It is acquired by specific uptake systems located in roots. Although the concentrations of K(+) in the soil solution are widely variable, K(+) nutrition is secured by uptake systems that exhibit different affinities for K(+). Two main systems have been described for root K(+) uptake in several species: the high-affinity HAK5-like transporter and the inward-rectifier AKT1-like channel. Other unidentified systems may be also involved in root K(+) uptake, although they only seem to operate when K(+) is not limiting. The use of knock-out lines has allowed demonstrating their role in root K(+) uptake in Arabidopsis and rice. Plant adaptation to the different K(+) supplies relies on the finely tuned regulation of these systems. Low K(+)-induced transcriptional up-regulation of the genes encoding HAK5-like transporters occurs through a signal cascade that includes changes in the membrane potential of root cells and increases in ethylene and reactive oxygen species concentrations. Activation of AKT1 channels occurs through phosphorylation by the CIPK23/CBL1 complex. Recently, activation of the Arabidopsis HAK5 by the same complex has been reported, pointing to CIPK23/CBL as a central regulator of the plant's adaptation to low K(+). Na(+) is not an essential plant nutrient but it may be beneficial for some plants. At low concentrations, Na(+) improves growth, especially under K(+) deficiency. Thus, high-affinity Na(+) uptake systems have been described that belong to the HKT and HAK families of transporters. At high concentrations, typical of saline environments, Na(+) accumulates in plant tissues at high concentrations, producing alterations that include toxicity, water deficit and K(+) deficiency. Data concerning pathways for Na(+) uptake into roots under saline conditions are still scarce, although several possibilities have been proposed. The apoplast is a significant pathway for Na(+) uptake in rice grown under salinity

Comparison between Arabidopsis and Rice for Main Pathways of K+ and Na+ Uptake by Roots

PubMed Central

Nieves-Cordones, Manuel; Martínez, Vicente; Benito, Begoña; Rubio, Francisco

2016-01-01

K+ is an essential macronutrient for plants. It is acquired by specific uptake systems located in roots. Although the concentrations of K+ in the soil solution are widely variable, K+ nutrition is secured by uptake systems that exhibit different affinities for K+. Two main systems have been described for root K+ uptake in several species: the high-affinity HAK5-like transporter and the inward-rectifier AKT1-like channel. Other unidentified systems may be also involved in root K+ uptake, although they only seem to operate when K+ is not limiting. The use of knock-out lines has allowed demonstrating their role in root K+ uptake in Arabidopsis and rice. Plant adaptation to the different K+ supplies relies on the finely tuned regulation of these systems. Low K+-induced transcriptional up-regulation of the genes encoding HAK5-like transporters occurs through a signal cascade that includes changes in the membrane potential of root cells and increases in ethylene and reactive oxygen species concentrations. Activation of AKT1 channels occurs through phosphorylation by the CIPK23/CBL1 complex. Recently, activation of the Arabidopsis HAK5 by the same complex has been reported, pointing to CIPK23/CBL as a central regulator of the plant’s adaptation to low K+. Na+ is not an essential plant nutrient but it may be beneficial for some plants. At low concentrations, Na+ improves growth, especially under K+ deficiency. Thus, high-affinity Na+ uptake systems have been described that belong to the HKT and HAK families of transporters. At high concentrations, typical of saline environments, Na+ accumulates in plant tissues at high concentrations, producing alterations that include toxicity, water deficit and K+ deficiency. Data concerning pathways for Na+ uptake into roots under saline conditions are still scarce, although several possibilities have been proposed. The apoplast is a significant pathway for Na+ uptake in rice grown under salinity conditions, but in other plant species

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

PubMed

Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

2016-01-01

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

PubMed

Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Cellular and molecular modifier pathways in tauopathies: the big picture from screening invertebrate models.

PubMed

Hannan, Shabab B; Dräger, Nina M; Rasse, Tobias M; Voigt, Aaron; Jahn, Thomas R

2016-04-01

Abnormal tau accumulations were observed and documented in post-mortem brains of patients affected by Alzheimer's disease (AD) long before the identification of mutations in the Microtubule-associated protein tau (MAPT) gene, encoding the tau protein, in a different neurodegenerative disease called Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). The discovery of mutations in the MAPT gene associated with FTDP-17 highlighted that dysfunctions in tau alone are sufficient to cause neurodegeneration. Invertebrate models have been diligently utilized in investigating tauopathies, contributing to the understanding of cellular and molecular pathways involved in disease etiology. An important discovery came with the demonstration that over-expression of human tau in Drosophila leads to premature mortality and neuronal dysfunction including neurodegeneration, recapitulating some key neuropathological features of the human disease. The simplicity of handling invertebrate models combined with the availability of a diverse range of experimental resources make these models, in particular Drosophila a powerful invertebrate screening tool. Consequently, several large-scale screens have been performed using Drosophila, to identify modifiers of tau toxicity. The screens have revealed not only common cellular and molecular pathways, but in some instances the same modifier has been independently identified in two or more screens suggesting a possible role for these modifiers in regulating tau toxicity. The purpose of this review is to discuss the genetic modifier screens on tauopathies performed in Drosophila and C. elegans models, and to highlight the common cellular and molecular pathways that have emerged from these studies. Here, we summarize results of tau toxicity screens providing mechanistic insights into pathological alterations in tauopathies. Key pathways or modifiers that have been identified are associated with a broad range of processes

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

NASA Astrophysics Data System (ADS)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, Ekhtear; Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp; Karnan, Sivasundaram

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, anmore » anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.« less

Stepwise pH-responsive nanoparticles for enhanced cellular uptake and on-demand intracellular release of doxorubicin.

PubMed

Chen, Wei-Liang; Li, Fang; Tang, Yan; Yang, Shu-di; Li, Ji-Zhao; Yuan, Zhi-Qiang; Liu, Yang; Zhou, Xiao-Feng; Liu, Chun; Zhang, Xue-Nong

2017-01-01

Physicochemical properties, including particle size, zeta potential, and drug release behavior, affect targeting efficiency, cellular uptake, and antitumor effect of nanocarriers in a formulated drug-delivery system. In this study, a novel stepwise pH-responsive nanodrug delivery system was developed to efficiently deliver and significantly promote the therapeutic effect of doxorubicin (DOX). The system comprised dimethylmaleic acid-chitosan-urocanic acid and elicited stepwise responses to extracellular and intracellular pH. The nanoparticles (NPs), which possessed negative surface charge under physiological conditions and an appropriate nanosize, exhibited advantageous stability during blood circulation and enhanced accumulation in tumor sites via enhanced permeability and retention effect. The tumor cellular uptake of DOX-loaded NPs was significantly promoted by the first-step pH response, wherein surface charge reversion of NPs from negative to positive was triggered by the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response in endo/lysosome acidic environment elicited the on-demand intracellular release of DOX from NPs, thereby increasing cytotoxicity against tumor cells. Furthermore, stepwise pH-responsive NPs showed enhanced antiproliferation effect and reduced systemic side effect in vivo. Hence, the stepwise pH-responsive NPs provide a promising strategy for efficient delivery of antitumor agents.

Stepwise pH-responsive nanoparticles for enhanced cellular uptake and on-demand intracellular release of doxorubicin

PubMed Central

Chen, Wei-liang; Li, Fang; Tang, Yan; Yang, Shu-di; Li, Ji-zhao; Yuan, Zhi-qiang; Liu, Yang; Zhou, Xiao-feng; Liu, Chun; Zhang, Xue-nong

2017-01-01

Physicochemical properties, including particle size, zeta potential, and drug release behavior, affect targeting efficiency, cellular uptake, and antitumor effect of nanocarriers in a formulated drug-delivery system. In this study, a novel stepwise pH-responsive nanodrug delivery system was developed to efficiently deliver and significantly promote the therapeutic effect of doxorubicin (DOX). The system comprised dimethylmaleic acid-chitosan-urocanic acid and elicited stepwise responses to extracellular and intracellular pH. The nanoparticles (NPs), which possessed negative surface charge under physiological conditions and an appropriate nanosize, exhibited advantageous stability during blood circulation and enhanced accumulation in tumor sites via enhanced permeability and retention effect. The tumor cellular uptake of DOX-loaded NPs was significantly promoted by the first-step pH response, wherein surface charge reversion of NPs from negative to positive was triggered by the slightly acidic tumor extracellular environment. After internalization into tumor cells, the second-step pH response in endo/lysosome acidic environment elicited the on-demand intracellular release of DOX from NPs, thereby increasing cytotoxicity against tumor cells. Furthermore, stepwise pH-responsive NPs showed enhanced antiproliferation effect and reduced systemic side effect in vivo. Hence, the stepwise pH-responsive NPs provide a promising strategy for efficient delivery of antitumor agents. PMID:28652730

Effects of Nanoparticle Size on Cellular Uptake and Liver MRI with PVP-Coated Iron Oxide Nanoparticles

PubMed Central

Huang, Jing; Bu, Lihong; Xie, Jin; Chen, Kai; Cheng, Zhen; Li, Xingguo; Chen, Xiaoyuan

2010-01-01

The effect of nanoparticle size (30–120 nm) on magnetic resonance imaging (MRI) of hepatic lesions in vivo has been systematically examined using polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles (PVP-IOs). Such biocompatible PVP-IOs with different sizes were synthesized by a simple one-pot pyrolysis method. These PVP-IOs exhibited good crystallinity and high T2 relaxivities, and the relaxivity increased with the size of the magnetic nanoparticles. It was found that cellular uptake changed with both size and surface physiochemical properties, and that PVP-IO-37 with a core size of 37 nm and hydrodynamic particle size of 100 nm exhibited higher cellular uptake rate and greater distribution than other PVP-IOs and Feridex. We systematically investigated the effect of nanoparticle size on MRI of normal liver and hepatic lesions in vivo. The physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their ability to accumulate in the liver. The contrast enhancement of PVP-IOs within the liver was highly dependent on the overall size of the nanoparticles, and the 100 nm PVP-IO-37 nanoparticles exhibited the greatest enhancement. These results will have implications in designing engineered nanoparticles that are optimized as MR contrast agents or for use in therapeutics. PMID:21043459

Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles

PubMed Central

Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

2014-01-01

Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles – GME-EC and GME-EC/MC nanoparticles – successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

Surface modification of solid lipid nanoparticles for oral delivery of curcumin: Improvement of bioavailability through enhanced cellular uptake, and lymphatic uptake.

PubMed

Baek, Jong-Suep; Cho, Cheong-Weon

2017-08-01

Curcumin has been reported to exhibit potent anticancer effects. However, poor solubility, bioavailability and stability of curcumin limit its in vivo efficacy for the cancer treatment. Solid lipid nanoparticles (SLN) are a promising delivery system for the enhancement of bioavailability of hydrophobic drugs. However, burst release of drug from SLN in acidic environment limits its usage as oral delivery system. Hence, we prepared N-carboxymethyl chitosan (NCC) coated curcumin-loaded SLN (NCC-SLN) to inhibit the rapid release of curcumin in acidic environment and enhance the bioavailability. The NCC-SLN exhibited suppressed burst release in simulated gastric fluid while sustained release was observed in simulated intestinal fluid. Furthermore, NCC-SLN exhibited increased cytotoxicity and cellular uptake on MCF-7 cells. The lymphatic uptake and oral bioavailability of NCC-SLN were found to be 6.3-fold and 9.5-fold higher than that of curcumin solution, respectively. These results suggest that NCC-SLN could be an efficient oral delivery system for curcumin. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco-2 Cells.

PubMed

Yang, Cheng; Zhang, Hua; Liu, Ronghua; Zhu, Honghui; Zhang, Lianfu; Tsao, Rong

2017-11-29

The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells. This study demonstrated that the trans-cis isomerization of all-E-astaxanthin and the cis-trans isomerization of Z-astaxanthins could happen both during in vitro gastrointestinal digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin in reported studies. All three astaxanthin isomers were effective in maintaining cellular redox homeostasis as seen in the antioxidant enzyme (CAT, SOD) activities ; 9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress as demonstrated by the lower cellular uptake of Z-astaxanthins and lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H 2 O 2 . We conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress induced intestinal diseases.

Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

PubMed Central

Lamichhane, Surya P; Arya, Neha; Ojha, Nirdesh; Kohler, Esther; Shastri, V Prasad

2015-01-01

The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 μg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%–60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy. PMID:25632234

Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction

NASA Astrophysics Data System (ADS)

Lu, Yi-Ching; Luo, Pei-Chun; Huang, Chun-Wan; Leu, Yann-Lii; Wang, Tzu-Hao; Wei, Kuo-Chen; Wang, Hsin-Ell; Ma, Yunn-Hwa

2014-08-01

Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3-gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1-3 μM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 °C, but not when EGCG was removed from the culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP-EGCG composite produced by adsorption of EGCG and magnetic separation also led to an enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system, which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.

On the mechanism of Cr (VI)-induced carcinogenesis: dose dependence of uptake and cellular responses.

PubMed

Liu, K; Husler, J; Ye, J; Leonard, S S; Cutler, D; Chen, F; Wang, S; Zhang, Z; Ding, M; Wang, L; Shi, X

2001-06-01

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

A polymeric nanoparticle consisting of mPEG-PLA-Toco and PLMA-COONa as a drug carrier: improvements in cellular uptake and biodistribution.

PubMed

Yi, Yilwoong; Kim, Jae Hong; Kang, Hye-Won; Oh, Hun Seung; Kim, Sung Wan; Seo, Min Hyo

2005-02-01

To evaluate a new polymeric nanoparticulate drug delivery formulation that consists of two components: i) an amphiphilic diblock copolymer having tocopherol moiety at the end of the hydrophobic block in which the hydrophobic tocopherol moiety increases stability of hydrophobic core of the nanoparticle in aqueous medium; and ii) a biodegradable copolyester having carboxylate end group that is capable of forming ionic complex with positively charged compounds such as doxorubicin. A doxourubicin-loaded polymeric nanoparticle (Dox-PNP) was prepared by solvent evaporation method. The entrapment efficiency, size distribution, and in vitro release profile at various pH conditions were characterized. In vitro cellular uptake was investigated by confocal microscopy, flow cytometry, and MTT assay using drug-sensitive and drug-resistant cell lines. Pharmacokinetics and biodistribution were evaluated in rats and tumor-bearing mice. Doxorubicin (Dox) was efficiently loaded into the PNP (higher than 95% of entrapment efficiency), and the diameter of Dox-PNP was in the range 20-25 nm with a narrow size distribution. In Vitro study showed that Dox-PNP exhibited higher cellular uptake into both human breast cancer cell (MCF-7) and human uterine cancer cell (MES-SA) than free doxorubicin solution (Free-Dox), especially into drug-resistant cells (MCF-7/ADR and MES-SA/Dx-5). In pharmacokinetics and tissue distribution study, the bioavailability of Dox-PNP calculated from the area under the blood concentration-time curve (AUC) was 69.8 times higher than that of Free-Dox in rats, and Dox-PNP exhibited 2 times higher bioavailability in tumor tissue of tumor-bearing mice. Dox-PNP exhibited enhanced cellular uptake of the drug. In the cytotoxic activity study, this improved cellular uptake was proved to be more advantageous in drug-resistant cell. Dox-PNP exhibited much higher bioavailability in blood plasma and more drug accumulation in tumor tissue than conventional doxorubicin

Human adenovirus Ad36 and its E4orf1 gene enhance cellular glucose uptake even in the presence of inflammatory cytokines.

PubMed

Na, Ha-Na; Dubuisson, Olga; Hegde, Vijay; Nam, Jae-Hwan; Dhurandhar, Nikhil V

2016-05-01

Aging and obesity are associated with elevated pro-inflammatory cytokines such as monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)α, which are linked to insulin resistance. Anti-inflammatory agents have marginal effect in improving insulin resistance. Hence, agents are needed to improve glycemic control despite the inflammation. Ad36, a human adenovirus, increases TNFα and MCP1 mRNA in adipose tissue, yet improves glycemic control in mice. Ad36 via its E4orf1 gene, up-regulates AKT/glucose transporter (Glut)-4 signaling to enhance cellular glucose uptake. Directly test a role of Ad36, or E4orf1 in enhancing cellular glucose uptake in presence of inflammatory cytokines. Experiment 1: 3T3-L1 preadipocytes were treated with 0, 10 or 100 ng/mL lipopolysaccharides (LPS), and infected with 0 or 5 plaque forming units (PFU) of Ad36/cell. 3T3-L1 cells that stably and inducibly express E4orf1 or a null vector (pTRE-E4orf1 or pTRE-null cells), were similarly treated with LPS and then with doxycycline, to induce E4orf1. Experiment 2: 3T3L1 preadipocytes were treated with 25 nM MCP1 or 20 nM TNFα for 16 h, followed by infection with 0 or 5 PFU of Ad36/cell. Experiment 3: pTRE-E4orf1 or -null cells were similarly treated with MCP1 or TNFα followed by doxycycline to induce E4orf1. Cellular glucose uptake and cellular signaling were determined 72 h post-Ad36 infection or E4orf1-induction, in continued presence of MCP1 or TNFα. In 3T3-L1 preadipocytes, Ad36, but not E4orf1, increased MCP1 and TNFα mRNA, in presence of LPS stimulation. Ad36 or E4orf1 up-regulated AKT-phosphorylation and Glut4 and increased glucose uptake (P < 0.05) in the presence of MCP1 or TNFα. Unlike Ad36, E4orf1 does not appear to stimulate inflammatory response. Ad36 and E4orf1 both enhance cellular glucose uptake even in presence of inflammation. Further research is needed to harness this novel and beneficial property of E4orf1 to improve hyperglycemia despite chronic

Cytotoxicity and cellular uptake of doxorubicin and its formamidine derivatives in HL60 sensitive and HL60/MX2 resistant cells.

PubMed

Kik, Krzysztof; Wasowska-Lukawska, Malgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-04-01

In this work a comparison was made of the cytotoxicity and cellular uptake of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N=CH-N<) at the 3' position with morpholine (DOXM) or hexamethyleneimine (DOXH) ring. All tests were performed in doxorubicin-sensitive HL60 and -resistant HL60/MX2 cells which are known for the presence of altered topoisomerase II. Cytotoxic activity of DOX toward HL60/MX2 cells was about 195 times lower when compared with the sensitive HL60 cell line. DOXM and DOXH were approximately 20 times more active in resistant cells than DOX. It was found that the uptake of DOX was lower in resistant cells by about 16%, while that of DOXM and DOXH was lower by about 36% and 19%, respectively. Thus the changes in the cellular uptake of anthracyclines are not associated with the fact that cytotoxicity of DOXM and DOXH exceed the cytotoxicity of DOX. Experiments in cell-free system containing human topoisomerase II showed that topoisomerase II is not inhibited by DOXM and DOXH. Formamidinoanthracyclines may be more useful than parent drugs in therapy against tumor cells with altered topoisomerase II activity.

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

PubMed

Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

2016-04-15

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

Cyanidin-3-rutinoside increases glucose uptake by activating the PI3K/Akt pathway in 3T3-L1 adipocytes.

PubMed

Choi, Kyung Ha; Lee, Hyun Ah; Park, Mi Hwa; Han, Ji-Sook

2017-09-01

In this study, the effect of cyanidin-3-rutinoside (C3R) on glucose uptake by 3T3-L1 adipocytes was studied. C3R significantly increased glucose uptake, which was associated with enhanced plasma membrane glucose transporter type 4 (PM-GLUT4) expression in 3T3-L1 adipocytes. The potentiating effect of C3R on glucose uptake and PM-GLUT4 expression was related to enhanced phosphorylation of insulin receptor substrate 1 (IRS-1) and Akt, as well as augmented activation of phosphatidylinositol-3-kinase (PI3K) in the insulin signaling pathway. C3R induced glucose uptake was inhibited only by the PI3K inhibitor, but not by an AMPK inhibitor in 3T3-L1 adipocytes. Therefore, C3R likely up-regulates glucose uptake and PM-GLUT4 expression in 3T3-L1 adipocytes by activating the PI3K/Akt pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation.

PubMed

Kubota, Kohei; Onishi, Kohei; Sawaki, Kazuaki; Li, Tianshu; Mitsuoka, Kaoru; Sato, Takaaki; Takeoka, Shinji

2017-01-01

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment.

Effect of the nanoformulation of siRNA-lipid assemblies on their cellular uptake and immune stimulation

PubMed Central

Kubota, Kohei; Onishi, Kohei; Sawaki, Kazuaki; Li, Tianshu; Mitsuoka, Kaoru; Sato, Takaaki; Takeoka, Shinji

2017-01-01

Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment. PMID:28790820

Tempo-spatially resolved cellular dynamics of human immunodeficiency virus transacting activator of transcription (Tat) peptide-modified nanocargos in living cells

NASA Astrophysics Data System (ADS)

Wei, Lin; Yang, Qiaoyu; Xiao, Lehui

2014-08-01

Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs

Visualization of self-delivering hydrophobically modified siRNA cellular internalization

PubMed Central

Ly, Socheata; Navaroli, Deanna M.; Didiot, Marie-Cécile; Cardia, James; Pandarinathan, Lakshmipathi; Alterman, Julia F.; Fogarty, Kevin; Standley, Clive; Lifshitz, Lawrence M.; Bellve, Karl D.; Prot, Matthieu; Echeverria, Dimas; Corvera, Silvia; Khvorova, Anastasia

2017-01-01

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention. PMID:27899655

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

PubMed

Petrova, Natalya S; Chernikov, Ivan V; Meschaninova, Mariya I; Dovydenko, Iiya S; Venyaminova, Aliya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

2012-03-01

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Inhibiting the NF-kappaB pathway to assess its function in the cellular response to space radiation

NASA Astrophysics Data System (ADS)

Koch, Kristina; Baumstark-Khan, Christa; Hellweg, Christine; Testard, Isabelle; Reitz, Guenther

2012-07-01

Radiation is regarded as one of the limiting factors for space missions. Therefore the cellular radiation response needs to be studied in order to estimate risks and to develop appropriate countermeasures. Exposure of human cells to ionizing radiation can provoke cell cycle arrest, leading to cellular senescence or premature differentiation, and different types of cell death. Previous heavy ion experiments have shown that the Nuclear Factor κB (NF-κB) pathway is activated by fluences that can be reached during long-term missions and thereby NF-κB was identified as an important modulating factor in the cellular radiation response. It could improve cellular survival after exposure to high radiation doses and influence the cancer risk of astronauts. The classical and the genotoxic stress induced NF-κB pathway result in nuclear translocation of the p65/p50 dimer. Both pathways might contribute to the cellular radiation response. Chemical inhibitors were tested to suppress the NF-κB pathway in recombinant HEK-pNF-κB-d2EGFP/Neo cells. The efficacy and cytotoxicity of the inhibitors targeting different elements of the NF-κB pathway were analyzed and found mostly inappropriate as inhibitors were partly cytotoxic or unspecific. Alternatively a functional knock-out of RelA (p65) was used to identify the contribution of the NF-κB pathway to different cellular outcomes. Small hairpin RNA constructs (shRNA) were transfected into the HEK-pNF-κB-d2EGFP/Neo cell line. Their functionality was assessed by quantitative Reverse Transcriptase real-time PCR (qRT-PCR) to verify that the RelA mRNA amount was reduced by more than 80% in the knock-down cells The original cell line had been stably transfected with a reporter system to monitor NF-κB activation by measuring destabilized Enhanced Green Fluorescent Protein (d2EGFP)-expression. It was shown that after 18 hours d2EGFP reaches its highest expression level after activation of NF-κB and can be measured by FACS analysis

Intracellular delivery of etoposide loaded biodegradable nanoparticles: cytotoxicity and cellular uptake studies.

PubMed

Yadav, Khushwant S; Jacob, Sheeba; Sachdeva, Geetanjali; Sawant, Krutika K

2011-08-01

The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGA-MPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 microM and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 microM for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.

Magnetic field enhanced cell uptake efficiency of magnetic silica mesoporous nanoparticles.

PubMed

Liu, Qian; Zhang, Jixi; Xia, Weiliang; Gu, Hongchen

2012-06-07

The advantages of using magnetic mesoporous silica nanoparticles (M-MSNs) in biomedical applications have been widely recognized. However, poor uptake efficiency may hinder the potential of M-MSNs in many applications, such as cell tracking, drug delivery, fluorescence and magnetic resonance imaging. An external magnetic field may improve the cellular uptake efficiency. In this paper, we evaluated the effect of a magnetic field on the uptake of M-MSNs. We found that the internalization of M-MSNs by A549 cancer cells could be accelerated and enhanced by a magnetic field. An endocytosis study indicated that M-MSNs were internalized by A549 cells mainly through an energy-dependent pathway, namely clathrin-induced endocytosis. Transmission electron microscopy showed that M-MSNs were trafficked into lysosomes. With the help of a magnetic field, anticancer drug-loaded M-MSNs induced elevated cancer cell growth inhibition.

Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast

PubMed Central

2011-01-01

Background The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. Results To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. Conclusions As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs. PMID:22023736

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum

PubMed Central

Zhao, Hua; Ruberu, Kalani; Li, Hongyun; Garner, Brett

2016-01-01

Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines. PMID:27893837

Adaptive Cellular Stress Pathways as Therapeutic Targets of Dietary Phytochemicals: Focus on the Nervous System

PubMed Central

Jo, Dong-Gyu; Park, Daeui; Chung, Hae Young

2014-01-01

During the past 5 decades, it has been widely promulgated that the chemicals in plants that are good for health act as direct scavengers of free radicals. Here we review evidence that favors a different hypothesis for the health benefits of plant consumption, namely, that some phytochemicals exert disease-preventive and therapeutic actions by engaging one or more adaptive cellular response pathways in cells. The evolutionary basis for the latter mechanism is grounded in the fact that plants produce natural antifeedant/noxious chemicals that discourage insects and other organisms from eating them. However, in the amounts typically consumed by humans, the phytochemicals activate one or more conserved adaptive cellular stress response pathways and thereby enhance the ability of cells to resist injury and disease. Examplesof such pathways include those involving the transcription factors nuclear factor erythroid 2-related factor 2, nuclear factor-κB, hypoxia-inducible factor 1α, peroxisome proliferator-activated receptor γ, and forkhead box subgroup O, as well as the production and action of trophic factors and hormones. Translational research to develop interventions that target these pathways may lead to new classes of therapeutic agents that act by stimulating adaptive stress response pathways to bolster endogenous defenses against tissue injury and disease. Because neurons are particularly sensitive to potentially noxious phytochemicals, we focus on the nervous system but also include findings from other cell types in which actions of phytochemicals on specific signal transduction pathways have been more thoroughly studied. PMID:24958636

The effect of tunicamycin on the glucose uptake, growth, and cellular adhesion in the protozoan parasite Crithidia fasciculata.

PubMed

Rojas, Robert; Segovia, Christopher; Trombert, Annette Nicole; Santander, Javier; Manque, Patricio

2014-10-01

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

PubMed Central

Jiang, Meiyan; Wang, Qi; Karasawa, Takatoshi; Koo, Ja-Won; Li, Hongzhe; Steyger, Peter S.

2014-01-01

Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2+/− mice, but not in Sglt2−/− mice. However, serum GTTR levels were elevated in Sglt2−/− mice compared to Sglt2+/− mice, and in phlorizin-treated Sglt2+/− mice compared to vehicle-treated Sglt2+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity. PMID:25268124

Cellular Delivery of Nanoparticles Revealed with Combined Optical and Isotopic Nanoscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proetto, Maria T.; Anderton, Christopher R.; Hu, Dehong

Synthetic drug-carrying nanomaterials offer great potential as targeted cellular delivery vehicles. Typically, their size, morphology, surface chemistry and stability are optimized in order to control their effect on drug release kinetics, cellular uptake pathways, efficiency and site of action. However, methods to track the carriers and their cargo independently at the micro- and nanoscale have been severely underutilized preventing the correlation between structure and function. Here we show that by using combined optical and isotopic nanoscopy we can track the uptake in cancer cells and subsequent drug release of a Pt(II)-loaded anticancer nanoparticle (NP) system. We found that by directlymore » polymerizing an oxaliplatin analogue containing a norbornyl moiety amenable to polymerization via ring opening metathesis polymerization (ROMP) we could generate amphiphiles in one pot. Spontaneous self-assembly of the drug-containing polymers in aqueous solution led to well-defined NPs in a reproducible manner. Our results demonstrate that the covalently loaded NPs are equipotent with free oxaliplatin and are taken up intact via endocytic pathways before release of the cytotoxic cargo. This was confirmed by super resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). We anticipate that this type of multimodal cellular tracking of NP and drug will bridge the knowledge gap between particle structure and performance for the vast array of currently generalizable systems in the literature. Furthermore, the use of covalently loaded NP drug systems should allow development of more stable, reproducible and site specific nanodelivery agents.« less

The influence of cellular uptake on gold nanorods photostability and photoacoustic conversion efficiency

NASA Astrophysics Data System (ADS)

Cavigli, Lucia; Ratto, Fulvio; Tatini, Francesca; Matteini, Paolo; Cini, Alberto; Giovannelli, Ilaria; de Angelis, Marella; Rossi, Francesca; Centi, Sonia; Pini, Roberto

2015-03-01

Their intense optical absorbance in the near-infrared window and chemical versatility make gold nanorods attractive for biomedical applications, such as photothermal therapies and photoacoustic imaging. However, their limited photostability remains a drawback of practical concern. In fact, when gold nanorods are irradiated with nanosecond laser pulses in resonance with their plasmon oscillations, there may occur reshaping into spherical particles or even fragmentation at higher optical fluences, which cause substantial modifications of their optical features with a loss of photoacoustic conversion efficiency. In this contribution, we focus on how the gold nanorods photostability is affected when these particles are modified for cellular uptake, by investigating their stability and photoacoustic conversion efficiency under near infrared pulsed irradiation at different laser fluences.

Cellular Uptake of the Clostridium perfringens Binary Iota-Toxin

PubMed Central

Blöcker, Dagmar; Behlke, Joachim; Aktories, Klaus; Barth, Holger

2001-01-01

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor. PMID:11292715

The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles.

PubMed

Smith, Carol-Anne M; de la Fuente, Jesus; Pelaz, Beatriz; Furlani, Edward P; Mullin, Margaret; Berry, Catherine C

2010-05-01

Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Activity of a sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and mechanism of ascorbate uptake

PubMed Central

Luo, Shuanghui; Wang, Zhiying; Kansara, Viral; Pal, Dhananjay; Mitra, Ashim. K.

2008-01-01

The objective of this research was to functionally characterize sodium-dependent vitamin C transporter (SVCT) in MDCK-MDR1 cells and to study the effect of substituted benzene derivatives on the intracellular accumulation of ascorbic acid (AA). Mechanism of AA uptake and transport was delineated. Uptake of [14C]ascorbic acid ([14C]AA) was studied in the absence and presence of excess unlabelled AA, anion transporter inhibitors, and a series of mono- and di- substituted benzenes. Transepithelial transport of [14C]AA across polarized cell membrane has been studied for the first time. Role of cellular protein kinase mediated pathways on the regulation of AA uptake has been investigated. The cellular localizations of SVCTs were observed using confocal microscopy. Uptake of AA was found to be saturable with a Km of 83.2 μM and Vmax of 94.2 pmol/min/mg protein for SVCT1. The process was pH, sodium, temperature, and energy dependent. It was under the regulation of cellular protein kinase C (PKC) and Ca2+/CaM mediated pathways. [14C]AA uptake was significantly inhibited in the presence of excess unlabelled AA and a series of electron-withdrawing group i.e. halogen- and nitro- substituted benzene derivatives. AA appears to translocate across polarized cell membrane from apical to basal side (A−B) as well as basal to apical side (B−A) at a similar permeability. It appears that SVCT1 was mainly expressed on the apical side and SVCT2 may be located on both apical and basal sides. In conclusion, SVCT has been functionally characterized in MDCK-MDR1 cells. The interference of a series of electrophile substituted benzenes on the AA uptake process may be explained by their structural similarity. SVCT may be targeted to facilitate the delivery of drugs with low bioavailability by conjugating with AA and its structural analogs. MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of AA conjugated prodrugs. PMID:18417304

Facile Synthesis of Uniform Virus-like Mesoporous Silica Nanoparticles for Enhanced Cellular Internalization

PubMed Central

2017-01-01

The low-efficiency cellular uptake property of current nanoparticles greatly restricts their application in the biomedical field. Herein, we demonstrate that novel virus-like mesoporous silica nanoparticles can easily be synthesized, showing greatly superior cellular uptake property. The unique virus-like mesoporous silica nanoparticles with a spiky tubular rough surface have been successfully synthesized via a novel single-micelle epitaxial growth approach in a low-concentration-surfactant oil/water biphase system. The virus-like nanoparticles’ rough surface morphology results mainly from the mesoporous silica nanotubes spontaneously grown via an epitaxial growth process. The obtained nanoparticles show uniform particle size and excellent monodispersity. The structural parameters of the nanoparticles can be well tuned with controllable core diameter (∼60–160 nm), tubular length (∼6–70 nm), and outer diameter (∼6–10 nm). Thanks to the biomimetic morphology, the virus-like nanoparticles show greatly superior cellular uptake property (invading living cells in large quantities within few minutes, <5 min), unique internalization pathways, and extended blood circulation duration (t1/2 = 2.16 h), which is much longer than that of conventional mesoporous silica nanoparticles (0.45 h). Furthermore, our epitaxial growth strategy can be applied to fabricate various virus-like mesoporous core–shell structures, paving the way toward designed synthesis of virus-like nanocomposites for biomedicine applications. PMID:28852697

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells.

PubMed

Lichtenstein, Dajana; Ebmeyer, Johanna; Knappe, Patrick; Juling, Sabine; Böhmert, Linda; Selve, Sören; Niemann, Birgit; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

2015-11-01

Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components--carbohydrates, proteins and fatty acids--were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides.

PubMed

Meade, Bryan R; Dowdy, Steven F

2008-03-01

The major limitation in utilizing information rich macromolecules for basic science and therapeutic applications is the inability of these large molecules to readily diffuse across the cellular membrane. While this restriction represents an efficient defense system against cellular penetration of unwanted foreign molecules and thus a crucial component of cell survival, overcoming this cellular characteristic for the intracellular delivery of macromolecules has been the focus of a large number of research groups worldwide. Recently, with the discovery of RNA interference, many of these groups have redirected their attention and have applied previously characterized cell delivery methodologies to synthetic short interfering RNA duplexes (siRNA). Protein transduction domain and cell penetrating peptides have been shown to enhance the delivery of multiple types of macromolecular cargo including peptides, proteins and antisense oligonucleotides and are now being utilized to enhance the cellular uptake of siRNA molecules. The dense cationic charge of these peptides that is critical for interaction with cell membrane components prior to internalization has also been shown to readily package siRNA molecules into stable nanoparticles that are capable of traversing the cell membrane. This review discusses the recent advances in noncovalent packaging of siRNA molecules with cationic peptides and the potential for the resulting complexes to successfully induce RNA interference within both in vitro and in vivo settings.

Effect of PEG molecular weight on stability, T₂ contrast, cytotoxicity, and cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs).

PubMed

Park, Yoonjee C; Smith, Jared B; Pham, Tuan; Whitaker, Ragnhild D; Sucato, Christopher A; Hamilton, James A; Bartolak-Suki, Elizabeth; Wong, Joyce Y

2014-07-01

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently unavailable as MRI contrast agents for detecting atherosclerosis in the clinical setting because of either low signal enhancement or safety concerns. Therefore, a new generation of SPIONs with increased circulation time, enhanced image contrast, and less cytotoxicity is essential. In this study, monodisperse SPIONs were synthesized and coated with polyethylene glycol (PEG) of varying molecular weights. The resulting PEGylated SPIONs were characterized, and their interactions with vascular smooth muscle cells (VSMCs) were examined. SPIONs were tested at different concentrations (100 and 500 ppm Fe) for stability, T2 contrast, cytotoxicity, and cellular uptake to determine an optimal formulation for in vivo use. We found that at 100 ppm Fe, the PEG 2K SPIONs showed adequate stability and magnetic contrast, and exhibited the least cytotoxicity and nonspecific cellular uptake. An increase in cell viability was observed when the SPION-treated cells were washed with PBS after 1h incubation compared to 5 and 24h incubation without washing. Our investigation provides insight into the potential safe application of SPIONs in the clinic. Copyright © 2014 Elsevier B.V. All rights reserved.

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling

PubMed Central

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V.

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling. PMID:27537838

E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling.

PubMed

Na, Ha-Na; Hegde, Vijay; Dubuisson, Olga; Dhurandhar, Nikhil V

2016-01-01

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling.

Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides.

PubMed

Allred, Benjamin E; Rupert, Peter B; Gauny, Stacey S; An, Dahlia D; Ralston, Corie Y; Sturzbecher-Hoehne, Manuel; Strong, Roland K; Abergel, Rebecca J

2015-08-18

Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.

Siderocalin-mediated recognition, sensitization, and cellular uptake of actinides

PubMed Central

Allred, Benjamin E.; Rupert, Peter B.; Gauny, Stacey S.; An, Dahlia D.; Ralston, Corie Y.; Sturzbecher-Hoehne, Manuel; Strong, Roland K.; Abergel, Rebecca J.

2015-01-01

Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin–transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein–ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications. PMID:26240330

Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability.

PubMed

Sun, Jiabei; Bi, Chao; Chan, Hok Man; Sun, Shaoping; Zhang, Qingwen; Zheng, Ying

2013-11-01

The aim of the present study was to blend liquid lipids with solid lipids to encapsulate curcumin in solid lipid nanoparticles (SLNs), thereby improving the dispersibility and chemical stability of curcumin, prolonging its antitumour activity and cellular uptake and enhancing its bioavailability. Curcumin-loaded SLNs (C-SLNs) were prepared by high-pressure homogenisation with liquid lipid Sefsol-218(®). The morphology, stability and release of curcumin in the optimised formulation were investigated. The anti-cancer activity of the formulation was evaluated in MCF-7 cells. Fluorescence spectrophotometry was used to quantify cellular uptake of the drug. The pharmacokinetic profiles of curcumin in SLNs after intravenous administration were studied in rats. Blending Sefsol-218(®) into a lipid matrix reduced the particle size without improving drug loading. An optimised formulation consisting of Dynasan 114(®), Sefsol-218(®), and Pluronic F68(®) (630:70:300, w/w) loaded with 0.8% drug was prepared. This formulation could be dispersed in water with a mean particle size of 152.8 ± 4.7 nm and a 90% entrapment efficiency. Curcumin displayed a two-phase sustained release profile from C-SLNs with improved chemical stability. Compared to the solubilised solution, C-SLNs exhibited prolonged inhibitory activity in cancer cells, as well as time-dependent increases in intracellular uptake. After intravenous administration to rats, the bioavailability of curcumin was increased by 1.25-fold. C-SLNs with improved dispersibility and chemical stability in an aqueous system have been successfully developed. C-SLNs may represent a potentially useful cancer therapeutic curcumin delivery system. Copyright © 2013 Elsevier B.V. All rights reserved.

The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function

PubMed Central

Padovano, Valeria; Kuo, Ivana Y.; Stavola, Lindsey K.; Aerni, Hans R.; Flaherty, Benjamin J.; Chapin, Hannah C.; Ma, Ming; Somlo, Stefan; Boletta, Alessandra; Ehrlich, Barbara E.; Rinehart, Jesse; Caplan, Michael J.

2017-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca2+ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O2 levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O2-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O2 and show less mitochondrial Ca2+ uptake in response to bradykinin-induced ER Ca2+ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O2 levels. PMID:27881662

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

PubMed

Halamoda-Kenzaoui, Blanka; Ceridono, Mara; Urbán, Patricia; Bogni, Alessia; Ponti, Jessica; Gioria, Sabrina; Kinsner-Ovaskainen, Agnieszka

2017-06-26

Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Cellular delivery of PEGylated PLGA nanoparticles.

PubMed

Pamujula, Sarala; Hazari, Sidhartha; Bolden, Gevoni; Graves, Richard A; Chinta, Dakshinamurthy Devanga; Dash, Srikanta; Kishore, Vimal; Mandal, Tarun K

2012-01-01

The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Co-Administration of an Excipient Oligonucleotide Helps Delineate Pathways of Productive and Nonproductive Uptake of Phosphorothioate Antisense Oligonucleotides in the Liver.

PubMed

Donner, Aaron J; Wancewicz, Edward V; Murray, Heather M; Greenlee, Sarah; Post, Noah; Bell, Melanie; Lima, Walt F; Swayze, Eric E; Seth, Punit P

2017-08-01

Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment. In this report, we measured PS ASO activity following administration by bolus, infusion or co-fusion with EON within hepatocytes and nonparenchymal cells (NPCs), of the liver. This revealed that while ASOs accumulate preferentially in NPCs, they are intrinsically more active in hepatocytes. Furthermore, we show that the EON enhances ASO potency when infused up to 72 h before or after administration of the active ASO suggesting that the EON can saturate and displace the ASO from nonproductive to productive compartments. Physical presence of the EON in tissues was required for optimal potentiation suggesting that there is a dynamic distribution of the ASO and EON between the compartments. Lastly, using a candidate approach, we confirmed Stabilin-2 as a molecular pathway for ASO uptake in sinusoidal endothelial cells and the ASGR as a pathway for ASO uptake into hepatocytes in the liver.

Nanodiamond internalization in cells and the cell uptake mechanism

NASA Astrophysics Data System (ADS)

Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

2013-08-01

Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

Magnetic field-enhanced cellular uptake of doxorubicin loaded magnetic nanoparticles for tumor treatment

NASA Astrophysics Data System (ADS)

Venugopal, Indu; Pernal, Sebastian; Duproz, Alexandra; Bentley, Jeromy; Engelhard, Herbert; Linninger, Andreas

2016-09-01

Cancer remains the second most common cause of death in the US, accounting for nearly 1 out of every 4 deaths. In recent years, several varieties of nanoparticles (NPs) have been synthesized with the intent of being utilized as tumor drug delivery vehicles. We have produced superparamagnetic, gold-coated magnetite (Fe3O4@Au) NPs and loaded them with the chemotherapeutic drug doxorubicin (DOX) for magnetic drug targeting (MDT) of tumors. The synthetic strategy uses the food thickening agent gellan gum (Phytagel) as a negatively charged shell around the Fe3O4@Au NP onto which the positively charged DOX molecules are loaded via electrostatic attraction. The resulting DOX-loaded magnetic nanoparticles (DOX-MNPs) were characterized using transmission electron microscopy, energy dispersive x-ray spectroscopy, superconducting quantum interference device magnetometry, surface area electron diffraction, zeta potential measurements, fourier transform infrared spectroscopy as well as UV/Vis and fluorescence spectroscopy. Cytotoxicity of the DOX-MNPs was demonstrated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on C6 glioma cells. Cellular uptake of DOX-MNPs was enhanced with magnetic fields, which was quantitatively determined using flow cytometry. This improved uptake also led to greater tumor cell death, which was measured using MTT assay. These MDT results are promising for a new therapy for cancer.

Antimetabolic Effects of Polyphenols in Breast Cancer Cells: Focus on Glucose Uptake and Metabolism.

PubMed

Keating, Elisa; Martel, Fátima

2018-01-01

In the last years, metabolic reprogramming became a new key hallmark of tumor cells. One of its components is a deviant energetic metabolism, known as Warburg effect-an aerobic lactatogenesis- characterized by elevated rates of glucose uptake and consumption with high-lactate production even in the presence of oxygen. Because many cancer cells display a greater sensitivity to glucose deprivation-induced cytotoxicity than normal cells, inhibitors of glucose cellular uptake (facilitative glucose transporter 1 inhibitors) and oxidative metabolism (glycolysis inhibitors) are potential therapeutic targets in cancer treatment. Polyphenols, abundantly contained in fruits and vegetables, are dietary components with an established protective role against cancer. Several molecular mechanisms are involved in the anticancer effect of polyphenols, including effects on apoptosis, cell cycle regulation, plasma membrane receptors, signaling pathways, and epigenetic mechanisms. Additionally, inhibition of glucose cellular uptake and metabolism in cancer cell lines has been described for several polyphenols, and this effect was shown to be associated with their anticarcinogenic effect. This work will review data showing an antimetabolic effect of polyphenols and its involvement in the chemopreventive/chemotherapeutic potential of these dietary compounds, in relation to breast cancer.

Effect of chirality on cellular uptake, imaging and photodynamic therapy of photosensitizers derived from chlorophyll-a.

PubMed

Srivatsan, Avinash; Pera, Paula; Joshi, Penny; Wang, Yanfang; Missert, Joseph R; Tracy, Erin C; Tabaczynski, Walter A; Yao, Rutao; Sajjad, Munawwar; Baumann, Heinz; Pandey, Ravindra K

2015-07-01

We have previously shown that the (124)I-analog of methyl 3-(1'-m-iodobenzyloxy) ethyl-3-devinyl-pyropheophorbide-a derived as racemic mixture from chlorophyll-a can be used for PET (positron emission tomography)-imaging in animal tumor models. On the other hand, as a non-radioactive analog, it showed excellent fluorescence and photodynamic therapy (PDT) efficacy. Thus, a single agent in a mixture of radioactive ((124)I-) and non-radioactive ((127)I) material can be used for both dual-imaging and PDT of cancer. Before advancing to Phase I human clinical trials, we evaluated the activity of the individual isomers as well as the impact of a chiral center at position-3(1) in directing in vitro/in vivo cellular uptake, intracellular localization, epithelial tumor cell-specific retention, fluorescence/PET imaging, and photosensitizing ability. The results indicate that both isomers (racemates), either as methyl ester or carboxylic acid, were equally effective. However, the methyl ester analogs, due to subcellular deposition into vesicular structures, were preferentially retained. All derivatives containing carboxylic acid at the position-17(2) were noted to be substrate for the ABCG2 (a member of the ATP binding cassette transporters) protein explaining their low retention in lung tumor cells expressing this transporter. The compounds in which the chirality at position-3 has been substituted by a non-chiral functionality showed reduced cellular uptake, retention and lower PDT efficacy in mice bearing murine Colon26 tumors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

PubMed

Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

2015-01-01

Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

Ursolic Acid Increases Glucose Uptake through the PI3K Signaling Pathway in Adipocytes

PubMed Central

He, Yonghan; Li, Wen; Li, Ying; Zhang, Shuocheng; Wang, Yanwen; Sun, Changhao

2014-01-01

Background Ursolic acid (UA), a triterpenoid compound, is reported to have a glucose-lowering effect. However, the mechanisms are not fully understood. Adipose tissue is one of peripheral tissues that collectively control the circulating glucose levels. Objective The objective of the present study was to determine the effect and further the mechanism of action of UA in adipocytes. Methods and Results The 3T3-L1 preadipocytes were induced to differentiate and treated with different concentrations of UA. NBD-fluorescent glucose was used as the tracer to measure glucose uptake and Western blotting used to determine the expression and activity of proteins involved in glucose transport. It was found that 2.5, 5 and 10 µM of UA promoted glucose uptake in a dose-dependent manner (17%, 29% and 35%, respectively). 10 µM UA-induced glucose uptake with insulin stimulation was completely blocked by the phosphatidylinositol (PI) 3-kinase (PI3K) inhibitor wortmannin (1 µM), but not by SB203580 (10 µM), the inhibitor of mitogen-activated protein kinase (MAPK), or compound C (2.5 µM), the inhibitor of AMP-activated kinase (AMPK) inhibitor. Furthmore, the downstream protein activities of the PI3K pathway, phosphoinositide-dependent kinase (PDK) and phosphoinositide-dependent serine/threoninekinase (AKT) were increased by 10 µM of UA in the presence of insulin. Interestingly, the activity of AS160 and protein kinase C (PKC) and the expression of glucose transporter 4 (GLUT4) were stimulated by 10 µM of UA under either the basal or insulin-stimulated status. Moreover, the translocation of GLUT4 from cytoplasm to cell membrane was increased by UA but decreased when the PI3K inhibitor was applied. Conclusions Our results suggest that UA stimulates glucose uptake in 3T3-L1 adipocytes through the PI3K pathway, providing important information regarding the mechanism of action of UA for its anti-diabetic effect. PMID:25329874

IGF-I enhances cellular senescence via the reactive oxygen species-p53 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handayaningsih, Anastasia-Evi; Takahashi, Michiko; Fukuoka, Hidenori

2012-08-24

Highlights: Black-Right-Pointing-Pointer Cellular senescence plays an important role in tumorigenesis and aging process. Black-Right-Pointing-Pointer We demonstrated IGF-I enhanced cellular senescence in primary confluent cells. Black-Right-Pointing-Pointer IGF-I enhanced cellular senescence in the ROS and p53-dependent manner. Black-Right-Pointing-Pointer These results may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging. -- Abstract: Cellular senescence is characterized by growth arrest, enlarged and flattened cell morphology, the expression of senescence-associated {beta}-galactosidase (SA-{beta}-gal), and by activation of tumor suppressor networks. Insulin-like growth factor-I (IGF-I) plays a critical role in cellular growth, proliferation, tumorigenesis, and regulation of aging. In the presentmore » study, we show that IGF-I enhances cellular senescence in mouse, rat, and human primary cells in the confluent state. IGF-I induced expression of a DNA damage marker, {gamma}H2AX, the increased levels of p53 and p21 proteins, and activated SA-{beta}-gal. In the confluent state, an altered downstream signaling of IGF-I receptor was observed. Treatment with a reactive oxygen species (ROS) scavenger, N-acetylcystein (NAC) significantly suppressed induction of these markers, indicating that ROS are involved in the induction of cellular senescence by IGF-I. In p53-null mouse embryonic fibroblasts, the IGF-I-induced augmentation of SA-{beta}-gal and p21 was inhibited, demonstrating that p53 is required for cellular senescence induced by IGF-I. Thus, these data reveal a novel pathway whereby IGF-I enhances cellular senescence in the ROS and p53-dependent manner and may explain the underlying mechanisms of IGF-I involvement in tumorigenesis and in regulation of aging.« less

Carbamylated low-density lipoprotein attenuates glucose uptake via a nitric oxide-mediated pathway in rat L6 skeletal muscle cells.

PubMed

Choi, Hye-Jung; Lee, Kyoung Jae; Hwang, Eun Ah; Mun, Kyo-Cheol; Ha, Eunyoung

2015-07-01

Carbamylation is a cyanate-mediated posttranslational modification. We previously reported that carbamylated low-density lipoprotein (cLDL) increases reactive oxygen species and apoptosis via a lectin-like oxidized LDL receptor mediated pathway in human umbilical vein endothelial cells. A recent study reported an association between cLDL and type 2 diabetes mellitus (T2DM). In the current study, the effects of cLDL on glucose transport were explored in skeletal muscle cells. The effect of cLDL on glucose uptake, glucose transporter 4 (GLUT4) translocation, and signaling pathway were examined in cultured rat L6 muscle cells using 2-deoxyglucose uptake, immunofluorescence staining and western blot analysis. The quantity of nitric oxide (NO) was evaluated by the Griess reaction. The effect of native LDL (nLDL) from patients with chronic renal failure (CRF-nLDL) on glucose uptake was also determined. It was observed that cLDL significantly attenuated glucose uptake and GLUT4 translocation to the membrane, which was mediated via the increase in inducible nitric oxide synthase (iNOS)-induced NO production. Tyrosine nitration of the insulin receptor substrate-1 (IRS‑1) was increased. It was demonstrated that CRF-nLDL markedly reduced glucose uptake compared with nLDL from healthy subjects. Collectively, these findings indicate that cLDL, alone, attenuates glucose uptake via NO-mediated tyrosine nitration of IRS‑1 in L6 rat muscle cells and suggests the possibility that cLDL is involved in the pathogenesis of T2DM.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Ibervillea sonorae (Cucurbitaceae) induces the glucose uptake in human adipocytes by activating a PI3K-independent pathway.

PubMed

Zapata-Bustos, Rocio; Alonso-Castro, Angel Josabad; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A

2014-03-28

Ibervillea sonorae (S. Watson) Greene (Cucurbitaceae), a plant used for the empirical treatment of type 2 diabetes in México, exerts antidiabetic effects on animal models but its mechanism of action remains unknown. The aim of this study is to investigate the antidiabetic mechanism of an Ibervillea sonorae aqueous extract (ISE). Non-toxic ISE concentrations were assayed on the glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes, both in the absence or the presence of insulin signaling pathway inhibitors, and on murine and human adipogenesis. Chemical composition of ISE was examined by spectrophotometric and HPLC techniques. ISE stimulated the 2-NBDGlucose uptake by mature adipocytes in a concentration-dependent manner. ISE 50 µg/ml induced the 2-NBDG uptake in insulin-sensitive 3T3-F442A, 3T3-L1 and human adipocytes by 100%, 63% and 33%, compared to insulin control. Inhibitors for the insulin receptor, PI3K, AKT and GLUT4 blocked the 2-NBDG uptake in murine cells, but human adipocytes were insensitive to the PI3K inhibitor Wortmannin. ISE 50 µg/ml also stimulated the 2-NBDG uptake in insulin-resistant adipocytes by 117% (3T3-F442A), 83% (3T3-L1) and 48% (human). ISE induced 3T3-F442A adipogenesis but lacked proadipogenic effects on 3T3-L1 and human preadipocytes. Chemical analyses showed the presence of phenolics in ISE, mainly an appreciable concentration of gallic acid. Ibervillea sonorae exerts its antidiabetic properties by means of hydrosoluble compounds stimulating the glucose uptake in human preadipocytes by a PI3K-independent pathway and without proadipogenic effects. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The effect of oil-water partition coefficient on the distribution and cellular uptake of liposome-encapsulated gold nanoparticles.

PubMed

Bao, Quan-Ying; Liu, Ai-Yun; Ma, Yu; Chen, Huan; Hong, Jin; Shen, Wen-Bin; Zhang, Can; Ding, Ya

2016-10-01

The shape, size, and surface features of nanoparticles greatly influence the structure and properties of resulting hybrid nanosystems. In this work, gold nanoparticles (GNPs) were modified via S-Au covalent bonding by glycol monomethyl ether thioctate with poly(ethylene glycol) methyl ether of different molecular weights (i.e., 350, 550, and 750Da). These modified GNPs (i.e., GNP350, GNP550, and GNP750) showed different oil-water partition coefficients (Kp), as detected using inductively coupled plasma-atomic emission spectroscopy. The different Kp values of the gold conjugates (i.e., 13.98, 2.11, and 0.036 for GNP350, GNP550, and GNP750, respectively) resulted in different conjugate localization within liposomes, as observed by transmission electron microscopy. In addition, the cellular uptake of hybrid liposomes co-encapsulating gold conjugates and Nile red was evaluated using intracellular fluorescence intensity. The results indicated that precise GNP localization in the hydrophilic or hydrophobic liposome cavity could be achieved by regulating the GNP oil-water partition coefficient via surface modification; such localization could further affect the properties and functions of hybrid liposomes, including their cellular uptake profiles. This study furthers the understanding not only of the interaction between liposomes and inorganic nanoparticles but also of adjusting liposome-gold hybrid nanostructure properties via the surface chemistry of gold materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of tripolyphosphate on cellular uptake and RNA interference efficiency of chitosan-based nanoparticles in Raw 264.7 macrophages.

PubMed

Xiao, Bo; Ma, Panpan; Ma, Lijun; Chen, Qiubing; Si, Xiaoying; Walter, Lewins; Merlin, Didier

2017-03-15

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that is mainly secreted by macrophages during inflammation. Here, we synthesized a series of N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chlorides (HTCCs), and then used a complex coacervation technique or tripolyphosphate (TPP)-assisted ionotropic gelation strategy to complex the HTCCs with TNF-α siRNA (siTNF) to form nanoparticles (NPs). The resultant NPs had a desirable particle size (210-279nm), a slightly positive zeta potential (14-22mV), and negligible cytotoxicity against Raw 264.7 macrophages and colon-26 cells. Subsequent cellular uptake tests demonstrated that the introduction of TPP to the NPs markedly increased their cellular uptake efficiency (to nearly 100%) compared with TPP-free NPs, and yielded a correspondingly higher intracellular concentration of siRNA. Critically, in vitro gene silencing experiments revealed that all of the TPP-containing NPs showed excellent efficiency in inhibiting the mRNA expression level of TNF-α (by approximately 85-92%, which was much higher than that obtained using Oligofectamine/siTNF complexes). Collectively, these results obviously suggest that our non-toxic TPP-containing chitosan-based NPs can be exploited as efficient siTNF carriers for the treatment of inflammatory diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Pathways of cellular proteostasis in aging and disease.

PubMed

Klaips, Courtney L; Jayaraj, Gopal Gunanathan; Hartl, F Ulrich

2018-01-02

Ensuring cellular protein homeostasis, or proteostasis, requires precise control of protein synthesis, folding, conformational maintenance, and degradation. A complex and adaptive proteostasis network coordinates these processes with molecular chaperones of different classes and their regulators functioning as major players. This network serves to ensure that cells have the proteins they need while minimizing misfolding or aggregation events that are hallmarks of age-associated proteinopathies, including neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It is now clear that the capacity of cells to maintain proteostasis undergoes a decline during aging, rendering the organism susceptible to these pathologies. Here we discuss the major proteostasis pathways in light of recent research suggesting that their age-dependent failure can both contribute to and result from disease. We consider different strategies to modulate proteostasis capacity, which may help develop urgently needed therapies for neurodegeneration and other age-dependent pathologies. © 2018 Klaips et al.

Coupled elasticity–diffusion model for the effects of cytoskeleton deformation on cellular uptake of cylindrical nanoparticles

PubMed Central

Wang, Jizeng; Li, Long

2015-01-01

Molecular dynamic simulations and experiments have recently demonstrated how cylindrical nanoparticles (CNPs) with large aspect ratios penetrate animal cells and inevitably deform cytoskeletons. Thus, a coupled elasticity–diffusion model was adopted to elucidate this interesting biological phenomenon by considering the effects of elastic deformations of cytoskeleton and membrane, ligand–receptor binding and receptor diffusion. The mechanism by which the binding energy drives the CNPs with different orientations to enter host cells was explored. This mechanism involved overcoming the resistance caused by cytoskeleton and membrane deformations and the change in configurational entropy of the ligand–receptor bonds and free receptors. Results showed that deformation of the cytoskeleton significantly influenced the engulfing process by effectively slowing down and even hindering the entry of the CNPs. Additionally, the engulfing depth was determined quantitatively. CNPs preferred or tended to vertically attack target cells until they were stuck in the cytoskeleton as implied by the speed of vertically oriented CNPs that showed much faster initial engulfing speeds than horizontally oriented CNPs. These results elucidated the most recent molecular dynamics simulations and experimental observations on the cellular uptake of carbon nanotubes and phagocytosis of filamentous Escherichia coli bacteria. The most efficient engulfment showed the stiffness-dependent optimal radius of the CNPs. Cytoskeleton stiffness exhibited more significant influence on the optimal sizes of the vertical uptake than the horizontal uptake. PMID:25411410

Acidity-promoted cellular uptake and drug release mediated by amine-functionalized block polycarbonates prepared via one-shot ring-opening copolymerization.

PubMed

Wang, Hua-Fen; Jia, Hui-Zhen; Chu, Yan-Feng; Feng, Jun; Zhang, Xian-Zheng; Zhuo, Ren-Xi

2014-04-01

This paper reports a drug nanovehicle self-assembled from an amine-functionalized block copolymer poly(6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione)-block-poly(1,3-dioxepan-2-one) (PADMC-b-PTeMC), which is prepared by controlable ring-opening block copolymerization attractively in a "one-shot feeding" pathway. The copolymers display high cell-biocompatibility with no apparent cytotoxicities detected in 293T and HeLa cells. Due to their amphiphilic nature, PADMC-b-PTeMC copolymers can self-assemble into nanosized micelles capable of loading anticancer drugs such as camptothecin (CPT) and doxorubicin (DOX). In particular, the outer PADMC shell endows the PADMC-b-PTeMC nanomicelles with pH-dependent control over the micellar morphology, cell uptake efficiency, and the drug release pattern. Confocal inspection reveals the remarkably enhanced cellular internalization of drug loaded micelles by cancerous HeLa cells at relatively lower pH 5.8 simulating the mildly acid microenvironment in tumors. Along with the acidity-triggered volume expansion of micelles, an accelerated CPT release in vitro occurs. The obtained results adumbrate the possibility of completely biodegradable PADMC-b-PTeMC as pH-sensitive drug carriers for tumor chemotherapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling the plant uptake of organic chemicals, including the soil-air-plant pathway.

PubMed

Collins, Chris D; Finnegan, Eilis

2010-02-01

The soil-air-plant pathway is potentially important in the vegetative accumulation of organic pollutants from contaminated soils. While a number of qualitative frameworks exist for the prediction of plant accumulation of organic chemicals by this pathway, there are few quantitative models that incorporate this pathway. The aim of the present study was to produce a model that included this pathway and could quantify its contribution to the total plant contamination for a range of organic pollutants. A new model was developed from three submodels for the processes controlling plant contamination via this pathway: aerial deposition, soil volatilization, and systemic translocation. Using the combined model, the soil-air-plant pathway was predicted to account for a significant proportion of the total shoot contamination for those compounds with log K(OA) > 9 and log K(AW) < -3. For those pollutants with log K(OA) < 9 and log K(AW) > -3 there was a higher deposition of pollutant via the soil-air-plant pathway than for those chemicals with log K(OA) > 9 and log K(AW) < -3, but this was an insignificant proportion of the total shoot contamination because of the higher mobility of these compounds via the soil-root-shoot pathway. The incorporation of the soil-air-plant pathway into the plant uptake model did not significantly improve the prediction of the contamination of vegetation from polluted soils when compared across a range of studies. This was a result of the high variability between the experimental studies where the bioconcentration factors varied by 2 orders of magnitude at an equivalent log K(OA). One potential reason for this is the background air concentration of the pollutants under study. It was found background air concentrations would dominate those from soil volatilization in many situations unless there was a soil hot spot of contamination, i.e., >100 mg kg(-1).

Cellular uptake and intracellular localization of poly (acrylic acid) nanoparticles in a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1.

PubMed

Felix, Lindsey C; Ortega, Van A; Goss, Greg G

2017-11-01

The ever-growing production of engineered nanoparticles (NPs) for use in many agricultural, commercial, consumer, and industrial applications will lead to their accidental or intentional release into the environment. Potential routes of environmental exposure include manufacturing or transport spills, disposal of NP-containing products down the drain and/or in landfills, as well as direct usage on agricultural land. Therefore, NPs will inevitably contaminate aquatic environments and interact with resident organisms. However, there is limited information regarding the mechanisms that regulate NP transport into fish from the environment. Thus, our primary objective was to elucidate the mechanism(s) underlying cellular uptake and intracellular fate of 3-9nm poly (acrylic acid) NPs loaded with the fluorescent dye Nile red using a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line (RTgill-W1). In vitro measurements with NP-treated RTgill-W1 cells were carried out using a combination of laser scanning confocal microscopy, flow cytometry, fluorescent biomarkers (transferrin, cholera toxin B subunit, and dextran), endocytosis inhibitors (chlorpromazine, genistein, and wortmannin), and stains (4', 6-diamidino-2-phenylindole, Hoechst 33342, CellMask Deep Red, and LysoTracker Yellow). Clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis and macropinocytosis pathways were active in RTgill-W1 cells, and these pathways were exploited by the non-cytotoxic NPs to enter these cells. We have demonstrated that NP uptake by RTgill-W1 cells was impeded when clathrin-coated pit formation was blocked by chlorpromazine. Furthermore, colocalization analysis revealed a moderate positive relationship between NPs and LysoTracker Yellow-positive lysosomal compartments indicating that CME was the dominant operative mechanism involved in NP internalization by RTgill-W1 cells. Overall, our results clearly show that fish gill epithelial cells internalized NPs via energy

Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy.

PubMed

Bhashyam, Siva; Fields, Anjali V; Patterson, Brandy; Testani, Jeffrey M; Chen, Li; Shen, You-Tang; Shannon, Richard P

2010-07-01

We have shown that glucagon-like peptide-1 (GLP-1[7-36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1-induced myocardial glucose uptake are unknown. Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7-36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20+/-7 nmol/min/g; DCM+GLP-1: 61+/-12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98+/-2 density units; DCM: 256+/-58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1-induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38alpha MAP kinase activity (DCM+vehicle: 97+/-22 pmol ATP/mg/min; DCM+GLP-1: 170+/-36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151+/-13 density units; DCM+GLP-1: 306+/-12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21+/-3% membrane bound; DCM+GLP-1: 39+/-3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38alpha MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine. GLP-1 stimulates myocardial glucose uptake through a non-Akt-1-dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Foliar uptake of radiocaesium from irrigation water by paddy rice (Oryza sativa): an overlooked pathway in contaminated environments.

PubMed

Uematsu, Shinichiro; Vandenhove, Hildegarde; Sweeck, Lieve; Hees, May Van; Wannijn, Jean; Smolders, Erik

2017-04-01

Flooded (paddy) rice (Oryza sativa) can take up ions from the irrigation water by foliar uptake via the exposed stem base. We hypothesised that the stem base uptake of radiocaesium (RCs) is a pathway for rice grown in RCs-contaminated environments. We developed a bi-compartmental device which discriminates the stem base from root RCs uptake from solutions, thereby using RCs isotopes ( 137 Cs and 134 Cs) with < 2% solution leak between the compartments. Radiocaesium uptake was linear over time (0-24 h). Radiocaesium uptake to the entire plant, expressed per dry weight of the exposed parts, was sixfold higher for the roots than for the exposed stem base. At equal RCs concentrations in both compartments, the exposed stem base and root uptake contributed almost equally to the total shoot RCs concentrations. Reducing potassium supply to the roots not only increased the root RCs uptake but also increased RCs uptake by the stem base. This study was the first to experimentally demonstrate active and internally regulated RCs uptake by the stem base of rice. Scenario calculations for the Fukushima-affected area predict that RCs in irrigation water could be an important source of RCs in rice as indirectly suggested from field data. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Quantitative assessment of surface functionality effects on microglial uptake and retention of PAMAM dendrimers

NASA Astrophysics Data System (ADS)

Liaw, Kevin; Gök, Ozgul; DeRidder, Louis B.; Kannan, Sujatha; Kannan, Rangaramanujam M.

2018-04-01

Dendrimers are a promising class of polymeric nanoparticles for delivery of therapeutics and diagnostics. Polyamidoamine (PAMAM) dendrimers have shown significant efficacy in many animal models, with performance dependent on surface functionalities. Understanding the effects of end groups on biological interactions is critical for rational design of dendrimer-mediated therapies. In this study, we quantify the cellular trafficking kinetics (endocytosis and exocytosis) of generation 4 neutral (D4-OH), cationic (D4-NH2), anionic (D3.5-COOH), and generation 6 neutral (D6-OH) PAMAM dendrimers to investigate the nanoscale effects of surface functionality and size on cellular interactions. Resting and LPS-activated microglia were studied due to their central roles in dendrimer therapies for central nervous system disorders. D4-OH exhibits greater cellular uptake and lower retention than the larger D6-OH. D4-OH and D3.5-COOH exhibit similar trafficking kinetics, while D4-NH2 exhibits significant membrane interactions, resulting in faster cell association but lower internalization. Cationic charge may also enhance vesicular escape for greater cellular retention and preferential partitioning to nuclei. LPS activation further improves uptake of dendrimers, with smaller and cationic dendrimers experiencing the greatest increases in uptake compared to resting microglia. These studies have implications for the dependence of trafficking pathway on dendrimer properties and inform the design of dendrimer constructs tailored to specific therapeutic needs. Cationic dendrimers are ideal for delivering genetic materials to nuclei, but toxicity may be a limiting factor. Smaller, neutral dendrimers are best suited for delivering high levels of therapeutics in acute neuroinflammation, while larger or cationic dendrimers provide robust retention for sustained release of therapeutics in longer-term diseases.

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

PubMed

Fuchs, Evelyn; Haas, Alexander K; Spooner, Robert A; Yoshimura, Shin-ichiro; Lord, J Michael; Barr, Francis A

2007-06-18

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

Effect of dispersants of multi-walled carbon nanotubes on cellular uptake and biological responses

PubMed Central

Haniu, Hisao; Saito, Naoto; Matsuda, Yoshikazu; Kim, Yoong-Ahm; Park, Ki Chul; Tsukahara, Tamotsu; Usui, Yuki; Aoki, Kaoru; Shimizu, Masayuki; Ogihara, Nobuhide; Hara, Kazuo; Takanashi, Seiji; Okamoto, Masanori; Ishigaki, Norio; Nakamura, Koichi; Kato, Hiroyuki

2011-01-01

Although there have been many reports about the cytotoxicity of multi-walled carbon nanotubes (MWCNTs), the results are still controversial. To investigate one possible reason, the authors investigated the influence of MWCNT dispersants on cellular uptake and cytotoxicity. Cytotoxicity was examined (measured by alamarBlue® assay), as well as intracellular MWCNT concentration and cytokine secretion (measured by flow cytometry) in human bronchial epithelial cells (BEAS-2B) exposed to a type of highly purified MWCNT vapor grown carbon fiber (VGCF®, Shōwa Denkō Kabushiki-gaisha, Tokyo, Japan) in three different dispersants (gelatin, carboxylmethyl cellulose, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The authors also researched the relationship between the intracellular concentration of MWCNTs and cytotoxicity by using two cell lines, BEAS-2B and MESO-1 human malignant pleural mesothelioma cells. The intracellular concentration of VGCF was different for each of the three dispersants, and the levels of cytotoxicity and inflammatory response were correlated with the intracellular concentration of VGCF. A relationship between the intracellular concentration of VGCF and cytotoxic effects was observed in both cell lines. The results indicate that dispersants affect VGCF uptake into cells and that cytotoxicity depends on the intracellular concentration of VGCF, not on the exposed dosage. Thus, toxicity appears to depend on exposure time, even at low VGCF concentrations, because VGCF is biopersistent. PMID:22228997

Coupled elasticity-diffusion model for the effects of cytoskeleton deformation on cellular uptake of cylindrical nanoparticles.

PubMed

Wang, Jizeng; Li, Long

2015-01-06

Molecular dynamic simulations and experiments have recently demonstrated how cylindrical nanoparticles (CNPs) with large aspect ratios penetrate animal cells and inevitably deform cytoskeletons. Thus, a coupled elasticity-diffusion model was adopted to elucidate this interesting biological phenomenon by considering the effects of elastic deformations of cytoskeleton and membrane, ligand-receptor binding and receptor diffusion. The mechanism by which the binding energy drives the CNPs with different orientations to enter host cells was explored. This mechanism involved overcoming the resistance caused by cytoskeleton and membrane deformations and the change in configurational entropy of the ligand-receptor bonds and free receptors. Results showed that deformation of the cytoskeleton significantly influenced the engulfing process by effectively slowing down and even hindering the entry of the CNPs. Additionally, the engulfing depth was determined quantitatively. CNPs preferred or tended to vertically attack target cells until they were stuck in the cytoskeleton as implied by the speed of vertically oriented CNPs that showed much faster initial engulfing speeds than horizontally oriented CNPs. These results elucidated the most recent molecular dynamics simulations and experimental observations on the cellular uptake of carbon nanotubes and phagocytosis of filamentous Escherichia coli bacteria. The most efficient engulfment showed the stiffness-dependent optimal radius of the CNPs. Cytoskeleton stiffness exhibited more significant influence on the optimal sizes of the vertical uptake than the horizontal uptake. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Phloretin attenuates hyperuricemia-induced endothelial dysfunction through co-inhibiting inflammation and GLUT9-mediated uric acid uptake.

PubMed

Liu, Shuyun; Yuan, Yujia; Zhou, Yijie; Zhao, Meng; Chen, Younan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

2017-10-01

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti-inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA-induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP-1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor-kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose-dependent manner. In contrast, phloretin significantly attenuated pro-inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor-kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA-induced endothelial injury via a synergic mechanism including direct anti-inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia-related cardiovascular diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

PubMed

Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

2016-01-01

Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells.

Improved cellular uptake of functionalized single-walled carbon nanotubes.

PubMed

Antonelli, A; Serafini, S; Menotta, M; Sfara, C; Pierigé, F; Giorgi, L; Ambrosi, G; Rossi, L; Magnani, M

2010-10-22

Single-walled carbon nanotubes (SWNTs) due to their unique structural and physicochemical properties, have been proposed as delivery systems for a variety of diagnostic and therapeutic agents. However, SWNTs have proven difficult to solubilize in aqueous solution, limiting their use in biological applications. In an attempt to improve SWNTs' solubility, biocompatibility, and to increase cell penetration we have thoroughly investigated the construction of carbon scaffolds coated with aliphatic carbon chains and phospholipids to obtain micelle-like structures. At first, oxidized SWNTs (2370 ± 30 nmol mg(-1) of SWNTs) were covalently coupled with an alcoholic chain (stearyl alcohol, C(18)H(37)OH; 816 nmol mg(-1) of SWNTs). Subsequently, SWNTs-COOC(18)H(37) derivatives were coated with phosphatidylethanolamine (PE) or -serine (PS) phospholipids obtaining micelle-like structures. We found that cellular uptake of these constructs by phagocytic cells occurs via an endocytotic mechanism for constructs larger than 400 nm while occurs via diffusion through the cell membrane for constructs up to 400 nm. The material that enters the cell by phagocytosis is actively internalized by macrophages and localizes inside endocytotic vesicles. In contrast the material that enters the cells by diffusion is found in the cell cytosol. In conclusion, we have realized new biomimetic constructs based on alkylated SWNTs coated with phospholipids that are efficiently internalized by different cell types only if their size is lower than 400 nm. These constructs are not toxic to the cells and could now be explored as delivery systems for non-permeant cargoes.

Anti-glioma activity and the mechanism of cellular uptake of asiatic acid-loaded solid lipid nanoparticles.

PubMed

Garanti, Tanem; Stasik, Aneta; Burrow, Andrea Julie; Alhnan, Mohamed A; Wan, Ka-Wai

2016-03-16

Asiatic acid (AA), a pentacyclic triterpene found in Centella Asiatica, has shown neuroprotective and anti-cancer activity against glioma. However, owing to its poor aqueous solubility, effective delivery and absorption across biological barriers, in particular the blood brain barrier (BBB), are challenging. Solid lipid nanoparticles (SLNs) have shown a promising potential as a drug delivery system to carry lipophilic drugs across the BBB, a major obstacle in brain cancer therapy. Nevertheless, limited information is available about the cytotoxic mechanisms of nano-lipidic carriers with AA on normal and glioma cells. This study assessed the anti-cancer efficacy of AA-loaded SLNs against glioblastoma and their cellular uptake mechanism in comparison with SVG P12 (human foetal glial) cells. SLNs were systematically investigated for three different solid lipids; glyceryl monostearate (MS), glyceryl distearate (DS) and glyceryl tristearate (TS). The non-drug containing MS-SLNs (E-MS-SLNs) did not show any apparent toxicity towards normal SVG P12 cells, whilst the AA-loaded MS-SLNs (AA-MS-SLNs) displayed a more favourable drug release profile and higher cytotoxicity towards U87 MG cells. Therefore, MS-SLNs were chosen for further in vitro studies. Cytotoxicity studies of SLNs (± AA) were performed using MTT assay where AA-SLNs showed significantly higher cytotoxicity towards U87 MG cells than SVG P12 normal cells, as confirmed by flow cell cytometry. Cellular uptake of SLNs also appeared to be preferentially facilitated by energy-dependent endocytosis as evidenced by fluorescence imaging and flow cell cytometry. Using the Annexin V-PI double staining technique, it was found that these AA-MS-SLNs displayed concentration-dependent apoptotic activity on glioma cells, which further confirms the potential of exploiting these AA-loaded MS-SLNs for brain cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Bumetanide hyperpolarizes Madin-Darby canine kidney cells and enhances cellular gentamicin uptake via elevating cytosolic Ca2+ thus facilitating intermediate conductance Ca2+-activated potassium channels

PubMed Central

Wang, Tian; Yang, Yu-qin; Karasawa, Takatoshi; Wang, Qi; Phillips, Amanda; Guan, Bing-Cai; Ma, Ke-Tao; Jiang, Meiyan; Xie, Ding-Hua; Steyger, Peter S.; Jiang, Zhi-Gen

2012-01-01

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby Canine kidney (MDCK) cells. We found that bumetanide and furosemide concentration-dependently enhanced cytoplasmic GTTR fluorescence by ~60%. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl− channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~−80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+]i, but not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current Vr near −85 mV, and reduced GTTR uptake by ~20%. La3+ alone hyperpolarized the cells by ~−14 mV, reduced the I/V slope with a net current Vr near −10 mV, and inhibited GTTR uptake by ~50%. In the presence of La3+, bumetanide caused negligible potential or I/V change. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating the intracellular Ca2+ and thus a facilitation of the

Bumetanide hyperpolarizes madin-darby canine kidney cells and enhances cellular gentamicin uptake by elevating cytosolic Ca(2+) thus facilitating intermediate conductance Ca(2+)--activated potassium channels.

PubMed

Wang, Tian; Yang, Yu-Qin; Karasawa, Takatoshi; Wang, Qi; Phillips, Amanda; Guan, Bing-Cai; Ma, Ke-Tao; Jiang, Meiyan; Xie, Ding-Hua; Steyger, Peter S; Jiang, Zhi-Gen

2013-04-01

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La(3+), a non-selective cation channel (NSCC) blocker, and by K(+) channel blockers Ba(2+) and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl(-) channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V r) ~-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba(2+) or clotrimazole, and absent in elevated [Ca(2+)]i, but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca(2+). Ba(2+) and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current V r near -85 mV, and reduced GTTR uptake by ~20 %. La(3+) alone hyperpolarized the cells by ~-14 mV, reduced the I/V slope with a net current V r near -10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La(3+), bumetanide-caused negligible change in potential or I/V. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca(2+) and thus facilitating

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

PubMed Central

Jiang, Yanxialei; Lee, Jeeyoung; Lee, Jung Hoon; Lee, Joon Won; Kim, Ji Hyeon; Choi, Won Hoon; Yoo, Young Dong; Cha-Molstad, Hyunjoo; Kim, Bo Yeon; Kwon, Yong Tae; Noh, Sue Ah; Kim, Kwang Pyo; Lee, Min Jae

2016-01-01

ABSTRACT The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux. PMID:27560450

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy.

PubMed

Misra, Santosh K; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-07-11

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C(3)-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C(3)-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C(3) with phospholipid was used to generate C(3)-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy

PubMed Central

Misra, Santosh K.; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-01-01

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C3-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C3-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C3 with phospholipid was used to generate C3-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies. PMID:27405011

Multi-functionality Redefined with Colloidal Carotene Carbon Nanoparticles for Synchronized Chemical Imaging, Enriched Cellular Uptake and Therapy

NASA Astrophysics Data System (ADS)

Misra, Santosh K.; Mukherjee, Prabuddha; Chang, Huei-Huei; Tiwari, Saumya; Gryka, Mark; Bhargava, Rohit; Pan, Dipanjan

2016-07-01

Typically, multiplexing high nanoparticle uptake, imaging, and therapy requires careful integration of three different functions of a multiscale molecular-particle assembly. Here, we present a simpler approach to multiplexing by utilizing one component of the system for multiple functions. Specifically, we successfully synthesized and characterized colloidal carotene carbon nanoparticle (C3-NP), in which a single functional molecule served a threefold purpose. First, the presence of carotene moieties promoted the passage of the particle through the cell membrane and into the cells. Second, the ligand acted as a potent detrimental moiety for cancer cells and, finally, the ligands produced optical contrast for robust microscopic detection in complex cellular environments. In comparative tests, C3-NP were found to provide effective intracellular delivery that enables both robust detection at cellular and tissue level and presents significant therapeutic potential without altering the mechanism of intracellular action of β-carotene. Surface coating of C3 with phospholipid was used to generate C3-Lipocoat nanoparticles with further improved function and biocompatibility, paving the path to eventual in vivo studies.

Towards magnetic-enhanced cellular uptake, MRI and chemotherapeutics delivery by magnetic mesoporous silica nanoparticles.

PubMed

Liu, Qian; Zhang, Jixi; Xia, Weiliang; Gu, Hongchen

2012-10-01

A type of nanoparticle with three functional modalities was prepared with the aim of providing a multifunctional drug delivery system. The nanoparticle was 50 nm in size, with 2.7 nm mesopores and a magnetic nanocrystal core, which was further doped with FITC to enable the tracking of cellular uptake. We demonstrated that the internalization of the nanoparticles in tumor cells could be enhanced by applying an external magnetic field and furthermore, this kind of nanoparticle could be used in magnetic targeted drug delivery. With high transverse relaxivity, the magnetic nanoparticles shortened proton relaxation time and induced high magnetic resonance imaging contrast in tumor cells. Studies on anticancer drug loading and delivery capacity of anticancer drugs also showed that this type of nanoparticles could load water-soluble doxorubicin, and produce a prominent inhibitive effect against tumor cells. Taken together, the presented nanoparticles could become a promising agent in cancer theranostics.

Cellular uptake and trafficking of polydiacetylene micelles

NASA Astrophysics Data System (ADS)

Gravel, Edmond; Thézé, Benoit; Jacques, Isabelle; Anilkumar, Parambath; Gombert, Karine; Ducongé, Frédéric; Doris, Eric

2013-02-01

Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells. Electronic supplementary information (ESI) available: Detailed synthetic procedures and supplementary figures. See DOI: 10.1039/c2nr34149b

Intracellular pathways following uptake of bevacizumab in RPE cells.

PubMed

Aboul Naga, Shereen Hassan; Dithmer, Michaela; Chitadze, Guranda; Kabelitz, Dieter; Lucius, Ralph; Roider, Johann; Klettner, Alexa

2015-02-01

The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Purification, immunotoxic effects, and cellular uptake of trichothecene mycotoxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witt, M.F.

1989-01-01

Studies were carried out to better understand how the trichothecenes alter immune function in animals and humans. Deoxynivalenol (DON) was purified for use in animal feeding studies. Dietary exposure to DON for 8 weeks altered the serum immunoglobulin profile in mice and decreased the splenic plaque-forming cell response to the antigen sheep red blood cells. The uptake of ({sup 3}H)T-2 toxin by a murine B-cell hybridoma was studied in order to learn more about the way in which trichothecenes interact with immune cells. A simple procedure was developed for the laboratory production and purification of gram quantities of crystalline DON.more » When Fusarium graminearum R6576 was grown on rice, concentrations of 600 to 700 ppm DON accumulated after 13 to 18 days of incubation. A DON derivative, 15-acetylDON, was also found at concentrations of 100 to 300 ppm after 7 to 10 days. DON was purified from crude culture extracts by water-saturated silica gel chromatography. Alpha-({sup 3}H)T-2 toxin of 99% chemical and radiochemical purity was prepared for use in uptake studies. Both the rate of uptake of ({sup 3}H)T-2 toxin by hybridomas and the time required for accumulation of ({sup 3}H)T-2 to reach equilibrium were proportional to the concentration of ({sup 3}H)T-2. ({sup 3}H)T-2 toxin accumulated by hybridomas was proportional to the concentration of ({sup 3}H)T-2 between 10{sup {minus}8} and 10{sup {minus}3} M. The rate of uptake of ({sup 3}H)jT-2 toxin by hybridomas was inhibited by the trichothecenes T-2 toxin, DON, verrucarin A, and roridin A, as well as the antibiotic anisomycin. The kinetics and concentration dependence of accumulation, along with the inhibition patterns, suggest that uptake of ({sup 3}H)T-2 toxin by hybridomas is mediated by binding of toxin to ribosomes.« less

Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells.

PubMed

Wittig, Anja; Gehrke, Helge; Del Favero, Giorgia; Fritz, Eva-Maria; Al-Rawi, Marco; Diabaté, Silvia; Weiss, Carsten; Sami, Haider; Ogris, Manfred; Marko, Doris

2017-01-13

Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways-both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration

Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species.

PubMed

Matulionyte, Marija; Dapkute, Dominyka; Budenaite, Laima; Jarockyte, Greta; Rotomskis, Ricardas

2017-02-10

In recent years, photoluminescent gold nanoclusters have attracted considerable interest in both fundamental biomedical research and practical applications. Due to their ultrasmall size, unique molecule-like optical properties, and facile synthesis gold nanoclusters have been considered very promising photoluminescent agents for biosensing, bioimaging, and targeted therapy. Yet, interaction of such ultra-small nanoclusters with cells and other biological objects remains poorly understood. Therefore, the assessment of the biocompatibility and potential toxicity of gold nanoclusters is of major importance before their clinical application. In this study, the cellular uptake, cytotoxicity, and intracellular generation of reactive oxygen species (ROS) of bovine serum albumin-encapsulated (BSA-Au NCs) and 2-(N-morpholino) ethanesulfonic acid (MES)capped photoluminescent gold nanoclusters (Au-MES NCs) were investigated. The results showed that BSA-Au NCs accumulate in cells in a similar manner as BSA alone, indicating an endocytotic uptake mechanism while ultrasmall Au-MES NCs were distributed homogeneously throughout the whole cell volume including cell nucleus. The cytotoxicity of BSA-Au NCs was negligible, demonstrating good biocompatibility of such BSA-protected Au NCs. In contrast, possibly due to ultrasmall size and thin coating layer, Au-MES NCs exhibited exposure time-dependent high cytotoxicity and higher reactivity which led to highly increased generation of reactive oxygen species. The results demonstrate the importance of the coating layer to biocompatibility and toxicity of ultrasmall photoluminescent gold nanoclusters.

Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species

PubMed Central

Matulionyte, Marija; Dapkute, Dominyka; Budenaite, Laima; Jarockyte, Greta; Rotomskis, Ricardas

2017-01-01

In recent years, photoluminescent gold nanoclusters have attracted considerable interest in both fundamental biomedical research and practical applications. Due to their ultrasmall size, unique molecule-like optical properties, and facile synthesis gold nanoclusters have been considered very promising photoluminescent agents for biosensing, bioimaging, and targeted therapy. Yet, interaction of such ultra-small nanoclusters with cells and other biological objects remains poorly understood. Therefore, the assessment of the biocompatibility and potential toxicity of gold nanoclusters is of major importance before their clinical application. In this study, the cellular uptake, cytotoxicity, and intracellular generation of reactive oxygen species (ROS) of bovine serum albumin-encapsulated (BSA-Au NCs) and 2-(N-morpholino) ethanesulfonic acid (MES)-capped photoluminescent gold nanoclusters (Au-MES NCs) were investigated. The results showed that BSA-Au NCs accumulate in cells in a similar manner as BSA alone, indicating an endocytotic uptake mechanism while ultrasmall Au-MES NCs were distributed homogeneously throughout the whole cell volume including cell nucleus. The cytotoxicity of BSA-Au NCs was negligible, demonstrating good biocompatibility of such BSA-protected Au NCs. In contrast, possibly due to ultrasmall size and thin coating layer, Au-MES NCs exhibited exposure time-dependent high cytotoxicity and higher reactivity which led to highly increased generation of reactive oxygen species. The results demonstrate the importance of the coating layer to biocompatibility and toxicity of ultrasmall photoluminescent gold nanoclusters. PMID:28208642

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties.

PubMed

Ahmad, Suhana; Zamry, Anes Ateqah; Tan, Hern-Tze Tina; Wong, Kah Keng; Lim, JitKang; Mohamud, Rohimah

2017-11-01

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthesis, characterisation, and in vitro cellular uptake kinetics of nanoprecipitated poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA) polymeric nanoparticle micelles for nanomedicine applications

NASA Astrophysics Data System (ADS)

Salvage, Jonathan P.; Smith, Tia; Lu, Tao; Sanghera, Amendeep; Standen, Guy; Tang, Yiqing; Lewis, Andrew L.

2016-10-01

Nanoscience offers the potential for great advances in medical technology and therapies in the form of nanomedicine. As such, developing controllable, predictable, and effective, nanoparticle-based therapeutic systems remains a significant challenge. Many polymer-based nanoparticle systems have been reported to date, but few harness materials with accepted biocompatibility. Phosphorylcholine (PC) based biomimetic materials have a long history of successful translation into effective commercial medical technologies. This study investigated the synthesis, characterisation, nanoprecipitation, and in vitro cellular uptake kinetics of PC-based polymeric nanoparticle micelles (PNM) formed by the biocompatible and pH responsive block copolymer poly(2-methacryloyloxyethyl phosphorylcholine)- b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA). Atom transfer radical polymerisation (ATRP), and gel permeation chromatography (GPC) were used to synthesise and characterise the well-defined MPC100-DPA100 polymer, revealing organic GPC, using evaporative light scatter detection, to be more accurate than aqueous GPC for this application. Subsequent nanoprecipitation investigations utilising photon correlation spectroscopy (PCS) revealed PNM size increased with polymer concentration, and conferred Cryo-stability. PNM diameters ranged from circa 64-69 nm, and increased upon hydrophobic compound loading, circa 65-71 nm, with loading efficiencies of circa 60 % achieved, whilst remaining monodisperse. In vitro studies demonstrated that the PNM were of low cellular toxicity, with colony formation and MTT assays, utilising V79 and 3T3 cells, yielding comparable results. Investigation of the in vitro cellular uptake kinetics revealed rapid, 1 h, cellular uptake of MPC100-DPA100 PNM delivered fluorescent probes, with fluorescence persistence for 48 h. This paper presents the first report of these novel findings, which highlight the potential of the system for nanomedicine application

Structural basis of lentiviral subversion of a cellular protein degradation pathway

NASA Astrophysics Data System (ADS)

Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

2014-01-01

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection.

PubMed

Palocci, Cleofe; Valletta, Alessio; Chronopoulou, Laura; Donati, Livia; Bramosanti, Marco; Brasili, Elisa; Baldan, Barbara; Pasqua, Gabriella

2017-12-01

PLGA NPs' cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs' size selection. In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500-600 nm.

Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway[C][W

PubMed Central

Hanzawa, Taiki; Shibasaki, Kyohei; Numata, Takahiro; Kawamura, Yukio; Gaude, Thierry; Rahman, Abidur

2013-01-01

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature. PMID:24003052

Cationic carbon quantum dots derived from alginate for gene delivery: One-step synthesis and cellular uptake.

PubMed

Zhou, Jie; Deng, Wenwen; Wang, Yan; Cao, Xia; Chen, Jingjing; Wang, Qiang; Xu, Wenqian; Du, Pan; Yu, Qingtong; Chen, Jiaxin; Spector, Myron; Yu, Jiangnan; Xu, Ximing

2016-09-15

Carbon quantum dots (CQDs), unlike semiconductor quantum dots, possess fine biocompatibility, excellent upconversion properties, high photostability and low toxicity. Here, we report multifunctional CQDs which were developed using alginate, 3% hydrogen peroxide and double distilled water through a facile, eco-friendly and inexpensive one-step hydrothermal carbonization route. In this reaction, the alginate served as both the carbon source and the cationization agent. The resulting CQDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties which could serve as an excellent DNA condensation. As non-viral gene vector being used for the first time, the CQDs showed considerably high transfection efficiency (comparable to Lipofectamine2000 and significantly higher than PEI, p<0.05) and negligible toxicity. The photoluminescence properties of CQDs also permitted easy tracking of the cellular-uptake. The findings showed that both caveolae- and clathrin-mediated endocytosis pathways were involved in the internalization process of CQDs/pDNA complexes. Taken together, the alginate-derived photoluminescent CQDs hold great potential in biomedical applications due to their dual role as efficient non-viral gene vectors and bioimaging probes. This manuscript describes a facile and simple one-step hydrothermal carbonization route for preparing optically tunable photoluminescent carbon quantum dots (CQDs) from a novel raw material, alginate. These CQDs enjoy low cytotoxicity, positive zeta potential, excellent ability to condense macromolecular DNA, and most importantly, notably high transfection efficiency. The interesting finding is that the negatively-charged alginate can convert into positively charged CQDs without adding any cationic reagents. The significance of this study is that the cationic carbon quantum dots play dual roles as both non-viral gene vectors and bioimaging probes at the same time, which are most desirable in many

Degradable self-assembling dendrons for gene delivery: experimental and theoretical insights into the barriers to cellular uptake.

PubMed

Barnard, Anna; Posocco, Paola; Pricl, Sabrina; Calderon, Marcelo; Haag, Rainer; Hwang, Mark E; Shum, Victor W T; Pack, Daniel W; Smith, David K

2011-12-21

This paper uses a combined experimental and theoretical approach to gain unique insight into gene delivery. We report the synthesis and investigation of a new family of second-generation dendrons with four triamine surface ligands capable of binding to DNA, degradable aliphatic-ester dendritic scaffolds, and hydrophobic units at their focal points. Dendron self-assembly significantly enhances DNA binding as monitored by a range of experimental methods and confirmed by multiscale modeling. Cellular uptake studies indicate that some of these dendrons are highly effective at transporting DNA into cells (ca. 10 times better than poly(ethyleneimine), PEI). However, levels of transgene expression are relatively low (ca. 10% of PEI). This indicates that these dendrons cannot navigate all of the intracellular barriers to gene delivery. The addition of chloroquine indicates that endosomal escape is not the limiting factor in this case, and it is shown, both experimentally and theoretically, that gene delivery can be correlated with the ability of the dendron assemblies to release DNA. Mass spectrometric assays demonstrate that the dendrons, as intended, do degrade under biologically relevant conditions over a period of hours. Multiscale modeling of degraded dendron structures suggests that complete dendron degradation would be required for DNA release. Importantly, in the presence of the lower pH associated with endosomes, or when bound to DNA, complete degradation of these dendrons becomes ineffective on the transfection time scale-we propose this explains the poor transfection performance of these dendrons. As such, this paper demonstrates that taking this kind of multidisciplinary approach can yield a fundamental insight into the way in which dendrons can navigate barriers to cellular uptake. Lessons learned from this work will inform future dendron design for enhanced gene delivery. © 2011 American Chemical Society

Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle.

PubMed

Lee, Yang; Fluckey, James D; Chakraborty, Sanjukta; Muthuchamy, Mariappan

2017-07-01

Insulin resistance is a well-known risk factor for obesity, metabolic syndrome (MetSyn) and associated cardiovascular diseases, but its mechanisms are undefined in the lymphatics. Mesenteric lymphatic vessels from MetSyn or LPS-injected rats exhibited impaired intrinsic contractile activity and associated inflammatory changes. Hence, we hypothesized that insulin resistance in lymphatic muscle cells (LMCs) affects cell bioenergetics and signaling pathways that consequently alter contractility. LMCs were treated with different concentrations of insulin or glucose or both at various time points to determine insulin resistance. Onset of insulin resistance significantly impaired glucose uptake, mitochondrial function, oxygen consumption rates, glycolysis, lactic acid, and ATP production in LMCs. Hyperglycemia and hyperinsulinemia also impaired the PI3K/Akt while enhancing the ERK/p38MAPK/JNK pathways in LMCs. Increased NF-κB nuclear translocation and macrophage chemoattractant protein-1 and VCAM-1 levels in insulin-resistant LMCs indicated activation of inflammatory mechanisms. In addition, increased phosphorylation of myosin light chain-20, a key regulator of lymphatic muscle contraction, was observed in insulin-resistant LMCs. Therefore, our data elucidate the mechanisms of insulin resistance in LMCs and provide the first evidence that hyperglycemia and hyperinsulinemia promote insulin resistance and impair lymphatic contractile status by reducing glucose uptake, altering cellular metabolic pathways, and activating inflammatory signaling cascades.-Lee, Y., Fluckey, J. D., Chakraborty, S., Muthuchamy, M. Hyperglycemia- and hyperinsulinemia-induced insulin resistance causes alterations in cellular bioenergetics and activation of inflammatory signaling in lymphatic muscle. © FASEB.

Functionalized single-walled carbon nanotubes: cellular uptake, biodistribution and applications in drug delivery.

PubMed

Li, Zixian; de Barros, Andre Luis Branco; Soares, Daniel Cristian Ferreira; Moss, Sara Nicole; Alisaraie, Laleh

2017-05-30

The unique properties of single-walled carbon nanotubes (SWNTs) enable them to play important roles in many fields. One of their functional roles is to transport cargo into cell. SWNTs are able to traverse amphipathic cell membranes due to their large surface area, flexible interactions with cargo, customizable dimensions, and surface chemistry. The cargoes delivered by SWNTs include peptides, proteins, nucleic acids, as well as drug molecules for therapeutic purpose. The drug delivery functions of SWNTs have been explored over the past decade. Many breakthrough studies have shown the high specificity and potency of functionalized SWNT-based drug delivery systems for the treatment of cancers and other diseases. In this review, we discuss different aspects of drug delivery by functionalized SWNT carriers, diving into the cellular uptake mechanisms, biodistribution of the delivery system, and safety concerns on degradation of the carriers. We emphasize the delivery of several common drugs to highlight the recent achievements of SWNT-based drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

Uptake of polycyclic aromatic hydrocarbons and their cellular effects in the mangrove Bruguiera gymnorrhiza.

PubMed

Naidoo, Gonasageran; Naidoo, Krishnaveni

2016-12-15

The uptake of polycyclic aromatic hydrocarbons and their cellular effects were investigated in the mangrove Bruguiera gymnorrhiza. Seedlings were subjected to sediment oiling for three weeks. In the oiled treatment, the ƩPAHs was higher in roots (99%) than in leaves (1%). In roots, PAHs included phenanthrene (55%), acenaphthene (13%), fluorine (12%) and anthracene (8%). In leaves, PAHs possessed two to three rings and included acenaphthene (35%), naphthalene (33%), fluorine (18%) and phenanthrene (14%). In the roots, oil caused disorganization of cells in the root cap, meristem and conducting tissue. Oil contaminated cells were distorted and possessed large and irregularly shaped vacuoles. Ultrastructural changes included loss of cell contents and fragmentation of the nucleus and mitochondrion. In the leaves, oil caused dilation and distortion of chloroplasts and disintegration of grana and lamellae. Oil targets critical organelles such as nuclei, chloroplasts and mitochondria which are responsible for cell vitality and energy transformation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of astaxanthin-loaded DNA/chitosan nanoparticles for improved cellular uptake and antioxidation capability.

PubMed

Wang, Qian; Zhao, Yingyuan; Guan, Lei; Zhang, Yaping; Dang, Qifeng; Dong, Ping; Li, Jing; Liang, Xingguo

2017-07-15

DNA/chitosan co-assemblies were initially used as nanocarriers for efficient astaxanthin encapsulation and delivery. The obtained astaxanthin-loaded DNA/chitosan (ADC) colloidal system was transparent and homogenous, with astaxanthin content up to 65μg/ml. Compared to free astaxanthin, ADC nanoparticles with an astaxanthin concentration as low as 3.35nM still showed a more powerful cytoprotective effect on H 2 O 2 -induced oxidative cell damage, and improved cell viability from 49.9% to 61.9%. The ROS scavenging efficiency of ADC nanoparticles was as high as 54.3%, which was 2-fold higher than that of free astaxanthin. Besides this, ADC nanoparticles were easily engulfed by Caco-2 cells in a short time, indicating that the encapsulated astaxanthin could be absorbed through endocytosis by intestinal epithelial cells. The improved antioxidation capability and facilitated cellular uptake enabled the ADC nanoparticles to be good candidates for efficient delivery and absorption of astaxanthin. Copyright © 2017 Elsevier Ltd. All rights reserved.

High frequency application of nanosecond pulsed electric fields alters cellular membrane disruption and fluorescent dye uptake

NASA Astrophysics Data System (ADS)

Steelman, Zachary A.; Tolstykh, Gleb P.; Beier, Hope T.; Ibey, Bennett L.

2016-03-01

Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a "threshold" point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

PubMed

Zaman, Raiyan T; Tuerkcan, Silvan; Mahmoudi, Morteza; Saito, Toshinobu; Yang, Phillip C; Chin, Frederick T; McConnell, Michael V; Xing, Lei

2018-01-01

Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 μm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/μm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/μm2, P = 0.003) and macrophages (0.430±0.023 fCi/μm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested

Camptothecin prodrug nanomicelle based on a boronate ester-linked diblock copolymer as the carrier of doxorubicin with enhanced cellular uptake.

PubMed

Gao, Ya; Xiao, Yi; Liu, Shiyuan; Yu, Jiahui

2018-02-01

A novel pH-sensitive polymeric prodrug of camptothecin (CPT) by polymerizing γ-camptothecin-glutamate N-carboxyanhydride (Glu (CPT)-NCA) on boronate ester-linked poly (ethyleneglycol) (PEG) directly via the amine-initiated ring open polymerization (ROP) has been developed. The resulting amphiphilic prodrug (mPEG-BC-PGluCPT) could self-assemble into nanoparticles and encapsulate doxorubicin (Dox) simultaneously in aqueous solution for dual-drug delivery. The formation of polymeric prodrug micelles (mPEG-BC@PGluCPT) was confirmed by the measurements of critical aggregation concentration (CAC), particle size, and morphology observations. The mPEG-BC@PGluCPT micelles were colloidally stable in solutions for two weeks. Polymeric prodrug micelles mPEG-BC@PGluCPT and Dox-loaded micelles mPEG-BC@PGluCPT⋅Dox showed sustained drug release profiles over 48 h. As expected, drug release was accelerated by the decreasement of pH value from 7.4 to 6.0, which demonstrated pH-dependent manner of drug release. Additionally, it was found that cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles on HepG2 cells was higher than that on HL-7702 cells, especially in culture medium at pH 6.0. The enhanced cellular uptake of mPEG-BC@PGluCPT⋅Dox micelles under acidic condition on HepG2 cells resulted in the higher cytotoxicity of mPEG-BC@PGluCPT⋅Dox micelles at acidic pH than that at pH 7.4.

Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

PubMed Central

Pérez-Escuredo, Jhudit; Dadhich, Rajesh K; Dhup, Suveera; Cacace, Andrea; Van Hée, Vincent F; De Saedeleer, Christophe J; Sboarina, Martina; Rodriguez, Fabien; Fontenille, Marie-Joséphine; Brisson, Lucie; Porporato, Paolo E; Sonveaux, Pierre

2016-01-01

ABSTRACT Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling. PMID:26636483

Dynamic cellular uptake of mixed-monolayer protected nanoparticles.

PubMed

Carney, Randy P; Carney, Tamara M; Mueller, Marie; Stellacci, Francesco

2012-12-01

Nanoparticles (NPs) are gaining increasing attention for potential application in medicine; consequently, studying their interaction with cells is of central importance. We found that both ligand arrangement and composition on gold nanoparticles play a crucial role in their cellular internalization. In our previous investigation, we showed that 66-34OT nanoparticles coated with stripe-like domains of hydrophobic (octanethiol, OT, 34%) and hydrophilic (11-mercaptoundecane sulfonate, MUS, 66%) ligands permeated through the cellular lipid bilayer via passive diffusion, in addition to endo-/pino-cytosis. Here, we show an analysis of NP internalization by DC2.4, 3T3, and HeLa cells at two temperatures and multiple time points. We study four NPs that differ in their surface structures and ligand compositions and report on their cellular internalization by intracellular fluorescence quantification. Using confocal laser scanning microscopy we have found that all three cell types internalize the 66-34OT NPs more than particles coated only with MUS, or particles coated with a very similar coating but lacking any detectable ligand shell structure, or 'striped' particles but with a different composition (34-66OT) at multiple data points.

Chirality-dependent cellular uptake of chiral nanocarriers and intracellular delivery of different amounts of guest molecules

NASA Astrophysics Data System (ADS)

Kehr, Nermin Seda; Jose, Joachim

2017-12-01

We demonstrate the organic molecules loaded and chiral polymers coated periodic mesoporous organosilica (PMO) to generate chiral nanocarriers that we used to study chirality-dependent cellular uptake in serum and serum-free media and the subsequent delivery of different amounts of organic molecules into cells. Our results show that the amount of internalized PMO and thus the transported amount of organic molecules by nanocarrier PMO into cells was chirality dependent and controlled by hard/soft protein corona formation on the PMO surfaces. Therefore, this study demonstrate that chiral porous nanocarriers could potentially be used as advanced drug delivery systems which are able to use the specific chiral surface-protein interactions to influence/control the amount of (bio)active molecules delivered to cells in drug delivery and/or imaging applications.

Cellular trafficking of low molecular weight heparin incorporated in layered double hydroxide nanoparticles in rat vascular smooth muscle cells.

PubMed

Gu, Zi; Rolfe, Barbara E; Thomas, Anita C; Campbell, Julie H; Lu, G Q Max; Xu, Zhi P

2011-10-01

This paper reports a clear elucidation of the pathway for the cellular delivery of layered double hydroxide (LDH) nanoparticles intercalated with anti-restenotic low molecular weight heparin (LMWH). Cellular uptake of LMWH-LDH conjugates into cultured rat vascular smooth muscle cells (SMCs) measured via flow cytometry was more than ten times greater than that of LMWH alone. Confocal and transmission electron microscopy showed LMWH-LDH conjugates taken up by endosomes, then released into the cytoplasm. We propose that LMWH-LDH is taken up via a unique 'modified endocytic' pathway, whereby the conjugate is internalized by SMCs in early endosomes, sorted in late endosomes, and quickly released from late endosomes/lysosomes, avoiding degradation. Treatment of cells with LMWH-LDH conjugates suppressed the activation of ERK1/2 in response to foetal calf serum (FCS) for up to 24h, unlike unconjugated LMWH which had no significant effect at 24h. Improved understanding of the intracellular pathway of LMWH-LDH nanohybrids in SMC will allow for refinement of design for LDH nanomedicine applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Novel theranostic zinc phthalocyanine-phospholipid complex self-assembled nanoparticles for imaging-guided targeted photodynamic treatment with controllable ROS production and shape-assisted enhanced cellular uptake.

PubMed

Ma, Jinyuan; Li, Yang; Liu, Guihua; Li, Ai; Chen, Yilin; Zhou, Xinyi; Chen, Dengyue; Hou, Zhenqing; Zhu, Xuan

2018-02-01

The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment. Copyright © 2017. Published by Elsevier B.V.

Scavenger receptor B1 facilitates macrophage uptake of silver nanoparticles and cellular activation

NASA Astrophysics Data System (ADS)

Aldossari, Abdullah A.; Shannahan, Jonathan H.; Podila, Ramakrishna; Brown, Jared M.

2015-07-01

Due to increased use of silver nanoparticles (AgNPs) for their antimicrobial activity, concerns have risen regarding potential adverse human health effects. Scavenger receptor B1 (SR-B1), a major receptor for high-density lipoprotein (HDL), is expressed by macrophages and has also been reported to play a role in recognition of negatively charged particles. We, therefore, hypothesized that SR-B1 mediates macrophage uptake of AgNPs and inflammatory activation. To test this hypothesis, we exposed a mouse macrophage cell line RAW264.7 (RAW) and bone marrow-derived macrophages (BMDM) to 20 nm citrate-suspended AgNPs. To verify the role of the SR-B1 receptor, we utilized a SR-B1 inhibitor (Blt2). In vitro studies demonstrated uptake of AgNPs and HDL-coated AgNPs by macrophages which were significantly reduced following pretreatment with Blt2. Inflammatory cytokine arrays revealed that macrophages exposed to AgNPs up-regulated expression of Tnf- α, Oncostatin m (OSM), Ccl4, Il17f, Ccl7, and Ccl2, whereas Il16 was found to be down-regulated. Macrophage activation was observed following AgNP and HDL-coated AgNP exposure as measured by OSM protein production and increased surface expression of CD86. These markers of activation were reduced with Blt2 pretreatment. The in vitro findings were confirmed in vivo through pulmonary instillation of AgNPs in mice. Pulmonary instillation of AgNPs resulted in a recruitment of inflammatory cells that were reduced in SR-B1-deficient mice or following Blt2 pretreatment. This study suggests that SR-B1 plays a major role in cellular recognition of AgNPs and the induction of cell responses that could contribute to inflammation caused by AgNP exposure.

Multifunctional organic–inorganic hybrid nanoparticles and nanosheets based on chitosan derivative and layered double hydroxide: cellular uptake mechanism and application for topical ocular drug delivery

PubMed Central

Chi, Huibo; Gu, Yan; Xu, Tingting; Cao, Feng

2017-01-01

To study the cellular uptake mechanism of multifunctional organic–inorganic hybrid nanoparticles and nanosheets, new chitosan–glutathione–valine–valine-layered double hydroxide (CG-VV-LDH) nanosheets with active targeting to peptide transporter-1 (PepT-1) were prepared, characterized and further compared with CG-VV-LDH nanoparticles. Both organic–inorganic hybrid nanoparticles and nanosheets showed a sustained release in vitro and prolonged precorneal retention time in vivo, but CG-VV-LDH nanoparticles showed superior permeability in the isolated cornea of rabbits than CG-VV-LDH nanosheets. Furthermore, results of cellular uptake on human corneal epithelial primary cells (HCEpiC) and retinal pigment epithelial (ARPE-19) cells indicated that both clathrin-mediated endocytosis and active transport of PepT-1 are involved in the internalization of CG-VV-LDH nanoparticles and CG-VV-LDH nanosheets. In summary, the CG-VV-LDH nanoparticle may be a promising carrier as a topical ocular drug delivery system for the treatment of ocular diseases of mid-posterior segments, while the CG-VV-LDH nanosheet may be suitable for the treatment of ocular surface diseases. PMID:28280329

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

PubMed

Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

2017-11-06

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

PubMed

Zheng, Nan; Lian, Bin; Du, Wenwen; Xu, Guobing; Ji, Jiafu

2018-01-01

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7). Copyright © 2017 Elsevier B.V. All rights reserved.

Oleic acid blocks EGF-induced [Ca2+]i release without altering cellular metabolism in fibroblast EGFR T17.

PubMed

Zugaza, J L; Casabiell, X A; Bokser, L; Casanueva, F F

1995-02-06

EGFR-T17 cells were pretreated with oleic acid and 5-10 minutes later stimulated with EGF, to study if early ionic signals are instrumental in inducing metabolic cellular response. Oleic acid blocks EGF-induced [Ca2+]i rise and Ca2+ influx without altering 2-deoxyglucose and 2-aminobutiryc acid uptake nor acute, nor chronically. Oleic acid it is shown, in the first minutes favors the entrance of both molecules to modify the physico-chemical membrane state. On the other hand, oleic acid is unable to block protein synthesis. The results suggest that EGF-induced Ins(1,4,5)P3/Ca2+ pathway does not seem to be decisive in the control of cellular metabolic activity.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2016-09-01

AWARD NUMBER: W81XWH-15-1-0420 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...Autosomal Dominant Polycystic Kidney Disease 5b. GRANT NUMBER W81XWH-15-1-0420 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kenneth R. Hallows, MD...polycystic kidney disease (ADPKD) is a common inherited disorder where patients, over the course of decades, develop large fluid filled cysts that

Surface Chemistry Manipulation of Gold Nanorods Displays High Cellular Uptake In Vitro While Preserving Optical Properties for Bio-Imaging and Photo-Thermal Applications

DTIC Science & Technology

2016-03-28

PROPERTIES FOR BIO -IMAGING AND PHOTO-THERMAL APPLICATIONS ANTHONY B. POLITO III, Maj, USAF, BSC, PhD, MT(ASCP)SBB March 2016 Final Report for March...HIGH CELLULAR UPTAKE IN VITRO WHILE PRESERVING OPTICAL PROPERTIES FOR BIO -IMAGING AND PHOTO-THERMAL APPLICATIONS. 5a. CONTRACT NUMBER 5b...These findings identify MTAB-TA GNRs as prime candidates for use in nano-based bio -imaging and photo-thermal applications. 15. SUBJECT TERMS

Aquaporins and root water uptake

USDA-ARS?s Scientific Manuscript database

Water is one of the most critical resources limiting plant growth and crop productivity, and root water uptake is an important aspect of plant physiology governing plant water use and stress tolerance. Pathways of root water uptake are complex and are affected by root structure and physiological res...

Industrial grade 2D molybdenum disulphide (MoS2): an in vitro exploration of the impact on cellular uptake, cytotoxicity, and inflammation

NASA Astrophysics Data System (ADS)

Moore, Caroline; Movia, Dania; Smith, Ronan J.; Hanlon, Damien; Lebre, Filipa; Lavelle, Ed C.; Byrne, Hugh J.; Coleman, Jonathan N.; Volkov, Yuri; McIntyre, Jennifer

2017-06-01

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and Live/Dead assays, it was established that 1 µg ml-1 (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the

HTLV Tax: A Fascinating Multifunctional Co-Regulator of Viral and Cellular Pathways

PubMed Central

Currer, Robert; Van Duyne, Rachel; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Das, Ravi; Narayanan, Aarthi; Kashanchi, Fatah

2012-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2–5% develop ATL. The past 35 years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-κB pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis. PMID:23226145

pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells.

PubMed

Yang, Mimi M; Wilson, William R; Wu, Zimei

2017-01-10

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6-2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL. Copyright © 2016 Elsevier B.V. All rights reserved.

Oxaloacetate- and acetoacetate-induced calcium efflux from mitochondria occurs by reversal of the uptake pathway.

PubMed Central

Bardsley, M E; Brand, M D

1982-01-01

1. Addition of oxaloacetate or acetoacetate to isolated rat liver mitochondria results in an efflux of Ca2+. Concomitant with this efflux is an immediate oxidation of endogenous nicotinamide nucleotides, a fall in the mitochondrial membrane potential and an increase in the rate of respiration. The primary effect in this sequence may be either (a) physiologically important stimulation of a Ca2+-efflux carrier, followed by Ca2+ re-uptake, a fall in membrane potential and increased respiration, or (b) physiologically unimportant damage to mitochondrial integrity, followed by a fall in membrane potential, increased respiration and Ca2+ efflux. 2. Ruthenium Red and EGTA will restore the increased respiratory rate to one approximating to the control rate of respiration. However, addition of lanthanide, at a concentration which inhibits the uptake but not the normal efflux of Ca2+, inhibits the rate of Ca2+ efflux induced by oxaloacetate or acetoacetate. Therefore the observed efflux is occurring by a reversal of the uptake pathway (uniporter) and thus follows the fall in membrane potential. 3. From these results we conclude that the decrease in membrane potential and increase in the rate of respiration seen during oxaloacetate- or acetoacetate-induced Ca2+ efflux cannot be accounted for by rapid Ca2+ cycling, but are due to damage to mitochondrial integrity. PMID:7082307

Cellular Homeostasis and Aging.

PubMed

Hartl, F Ulrich

2016-06-02

Aging and longevity are controlled by a multiplicity of molecular and cellular signaling events that interface with environmental factors to maintain cellular homeostasis. Modulation of these pathways to extend life span, including insulin-like signaling and the response to dietary restriction, identified the cellular machineries and networks of protein homeostasis (proteostasis) and stress resistance pathways as critical players in the aging process. A decline of proteostasis capacity during aging leads to dysfunction of specific cell types and tissues, rendering the organism susceptible to a range of chronic diseases. This volume of the Annual Review of Biochemistry contains a set of two reviews addressing our current understanding of the molecular mechanisms underlying aging in model organisms and humans.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2016-09-01

AWARD NUMBER: W81XWH-15-1-0419 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...Polycystic Kidney Disease 5b. GRANT NUMBER W81XWH-15-1-0419 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Michael J. Caplan, MD, PhD Kenneth...dominant polycystic kidney disease (ADPKD) is a common inherited disorder where patients, over the course of decades, develop large fluid filled

Preparation of Deep Sea Fish Oil-Based Nanostructured Lipid Carriers with Enhanced Cellular Uptake.

PubMed

Zhu, Qiu-Yun; Guissi, Fida; Yang, Ru-Ya; Wang, Qian; Wang, Ke; Chen, Dan; Han, Zhi-Hao; Ma, Yi; Zhang, Min; Gu, Yue-Qing

2015-12-01

Nanostructured lipid carriers (NLC) are a promising pharmaceutical delivery system with mean diameter less than 200 nm which are dispersed in an aqueous phase containing emulsifier(s), to increase the water solubility, stability and bioavailability of oil compounds. Herein we prepared a promising NLC with glyceryl monostearate (GMS) as the solid lipid template and deep sea fish oil as the liquid lipid template using melted-ultrasonic method. Fish oil-NLC had a mean size of 84.7 ± 2.6 nm and a zeta potential that ranged from -17.87 mV to -32.91 mV. The nanoparticles exhibited good stability for four weeks with a high encapsulation efficiency of 87.5 ± 5.2%. Afterwards, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to investigate the contribution of Fish oil-NLC in enhancing fluorescein isothiocyanate (FITC) cellular uptake in comparison with free FITC. The results of this study indicated the possibility of this carrier to overcome the shortcomings of deep sea fish oil and to provide a novel bifunctional carrier with nutritional potential and drug delivery ability.

Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo.

PubMed

Anand, Preetha; Nair, Hareesh B; Sung, Bokyung; Kunnumakkara, Ajaikumar B; Yadav, Vivek R; Tekmal, Rajeshwar R; Aggarwal, Bharat B

2010-02-01

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with antioxidant, anti-inflammatory, antiproliferative, anticancer, antidiabetic, antirheumatic, and antiviral effects, but its optimum potential is limited by its lack of solubility in aqueous solvents and poor oral bioavailability. We employed a polymer-based nanoparticle approach to improve bioavailability. Curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy indicated a particle diameter of 80.9 nm. This curcumin, renamed from hereon "as curcumin (NP)", was characterized for its biological activity. In vitro curcumin (NP) exhibited very rapid and more efficient cellular uptake than curcumin. Estrase staining revealed that curcumin (NP) was at least as potent as or more potent than curcumin in inducing apoptosis of leukemic cells and in suppressing proliferation of various tumor cell lines. When examined by electrophoretic gel shift mobility assay, curcumin (NP) was more active than curcumin in inhibiting TNF-induced NF-kappaB activation and in suppression of NF-kappaB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). In mice, curcumin (NP) was more bioavailable and had a longer half-life than curcumin. Overall we demonstrate that curcumin-loaded PLGA nanoparticles formulation has enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo over curcumin.

Gold(I) NHC Complexes: Antiproliferative Activity, Cellular Uptake, Inhibition of Mammalian and Bacterial Thioredoxin Reductases, and Gram-Positive Directed Antibacterial Effects.

PubMed

Schmidt, Claudia; Karge, Bianka; Misgeld, Rainer; Prokop, Aram; Franke, Raimo; Brönstrup, Mark; Ott, Ingo

2017-02-03

Gold complexes with N-heterocyclic carbene (NHC) ligands represent a promising class of metallodrugs for the treatment of cancer or infectious diseases. In this report, the synthesis and the biological evaluation of halogen-containing NHC-Au I -Cl complexes are described. The complexes 1 and 5 a-5 f displayed good cytotoxic activity against tumor cells, and cellular uptake studies suggested that an intact Au-NHC fragment is essential for the accumulation of high amounts of both the metal and the NHC ligand. However, the bioavailability was negatively affected by serum components of the cell culture media and was influenced by likely transformations of the complex. One example (5 d) efficiently induced apoptosis in vincristine- and daunorubicin-resistant P-glycoprotein overexpressing Nalm-6 leukemia cells. Cellular uptake studies with this compound showed that both the wild-type and resistant Nalm-6 cells accumulated comparable amounts of gold, indicating that the gold drug was not excreted by P-glycoprotein or other efflux transporters. The effective inhibition of mammalian and bacterial thioredoxin reductases (TrxR) was confirmed for all of the gold complexes. Antibacterial screening of the gold complexes showed a particularly high activity against Gram-positive strains, reflecting their high dependence on an intact Trx/TrxR system. This result is of particular interest as the inhibition of bacterial TrxR represents a relatively little explored mechanism of new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uptake of gentamicin by separated, viable renal tubules from rabbits.

PubMed

Barza, M; Murray, T; Hamburger, R J

1980-04-01

The proximal renal tubules have a marked affinity for gentamicin; they also are the major site of nephrotoxicity caused by this drug. The uptake of radiolabeled gentamicin in separated, viable renal tubules prepared by enzymatic digestion of rabbit kidneys was studied. The preparations showed rapid initial uptake of gentamicin followed by continued slower uptake. Accumulation was not affected by pH, but was significantly inhibited by ouabain, dinitrophenol, anoxia, and hypothermia in the absence of evident cellular damage. At gentamicin concentrations of greater than 50 microgram/ml in the medium, there was competition for drug uptake. Gentamicin efflux in tubules that were taken from a medium containing antibiotic and placed into antibiotic-free fluid was slow and incomplete. From these data it appears that gentamicin uptake by separated renal tubules occurs by a process that requires metabolic energy; thereafter, the drug resides in a poorly exchangeable cellular pool.

Distinct pathways leading to TDP-43-induced cellular dysfunctions.

PubMed

Yamashita, Makiko; Nonaka, Takashi; Hirai, Shinobu; Miwa, Akiko; Okado, Haruo; Arai, Tetsuaki; Hosokawa, Masato; Akiyama, Haruhiko; Hasegawa, Masato

2014-08-15

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component protein of inclusions found in brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the molecular mechanisms by which TDP-43 causes neuronal dysfunction and death remain unknown. Here, we report distinct cytotoxic effects of full-length TDP-43 (FL-TDP) and its C-terminal fragment (CTF) in SH-SY5Y cells. When FL-TDP was overexpressed in the cells using a lentiviral system, exogenous TDP-43, like endogenous TDP-43, was expressed mainly in nuclei of cells without any intracellular inclusions. However, these cells showed striking cell death, caspase activation and growth arrest at G2/M phase, indicating that even simple overexpression of TDP-43 induces cellular dysfunctions leading to apoptosis. On the other hand, cells expressing TDP-43 CTF showed cytoplasmic aggregates but without significant cell death, compared with cells expressing FL-TDP. Confocal microscopic analyses revealed that RNA polymerase II (RNA pol II) and several transcription factors, such as specificity protein 1 and cAMP-response-element-binding protein, were co-localized with the aggregates of TDP-43 CTF, suggesting that sequestration of these factors into TDP-43 aggregates caused transcriptional dysregulation. Indeed, accumulation of RNA pol II at TDP-43 inclusions was detected in brains of patients with FTLD-TDP. Furthermore, apoptosis was not observed in affected neurons of FTLD-TDP brains containing phosphorylated and aggregated TDP-43 pathology. Our results suggest that different pathways of TDP-43-induced cellular dysfunction may contribute to the degeneration cascades involved in the onset of ALS and FTLD-TDP. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cellular membrane trafficking of mesoporous silica nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, I-Ju

This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulfmore » some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to

Uptake of gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein by cultured rat granulosa cells: cellular mechanisms involved in lipoprotein metabolism and their importance to steroidogenesis

PubMed Central

1985-01-01

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold- LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34- 38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold- LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold- labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL- cholesterol vital for steroidogenesis. PMID:3920223

SPIKE – a database, visualization and analysis tool of cellular signaling pathways

PubMed Central

Elkon, Ran; Vesterman, Rita; Amit, Nira; Ulitsky, Igor; Zohar, Idan; Weisz, Mali; Mass, Gilad; Orlev, Nir; Sternberg, Giora; Blekhman, Ran; Assa, Jackie; Shiloh, Yosef; Shamir, Ron

2008-01-01

Background Biological signaling pathways that govern cellular physiology form an intricate web of tightly regulated interlocking processes. Data on these regulatory networks are accumulating at an unprecedented pace. The assimilation, visualization and interpretation of these data have become a major challenge in biological research, and once met, will greatly boost our ability to understand cell functioning on a systems level. Results To cope with this challenge, we are developing the SPIKE knowledge-base of signaling pathways. SPIKE contains three main software components: 1) A database (DB) of biological signaling pathways. Carefully curated information from the literature and data from large public sources constitute distinct tiers of the DB. 2) A visualization package that allows interactive graphic representations of regulatory interactions stored in the DB and superposition of functional genomic and proteomic data on the maps. 3) An algorithmic inference engine that analyzes the networks for novel functional interplays between network components. SPIKE is designed and implemented as a community tool and therefore provides a user-friendly interface that allows registered users to upload data to SPIKE DB. Our vision is that the DB will be populated by a distributed and highly collaborative effort undertaken by multiple groups in the research community, where each group contributes data in its field of expertise. Conclusion The integrated capabilities of SPIKE make it a powerful platform for the analysis of signaling networks and the integration of knowledge on such networks with omics data. PMID:18289391

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

PubMed

Fiorentino, Ilaria; Gualtieri, Roberto; Barbato, Vincenza; Mollo, Valentina; Braun, Sabrina; Angrisani, Alberto; Turano, Mimmo; Furia, Maria; Netti, Paolo A; Guarnieri, Daniela; Fusco, Sabato; Talevi, Riccardo

2015-01-15

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

NASA Astrophysics Data System (ADS)

Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

2016-02-01

Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids.

PubMed

Khalifeh-Soltani, Amin; McKleroy, William; Sakuma, Stephen; Cheung, Yuk Yin; Tharp, Kevin; Qiu, Yifu; Turner, Scott M; Chawla, Ajay; Stahl, Andreas; Atabai, Kamran

2014-02-01

Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8(-/-)) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through αvβ3 integrin- and αvβ5 integrin-dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.

Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

PubMed

Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2014-01-01

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

Measles Virus Enters Breast and Colon Cancer Cell Lines through a PVRL4-Mediated Macropinocytosis Pathway

PubMed Central

Delpeut, Sebastien; Sisson, Gary; Black, Karen M.

2017-01-01

ABSTRACT Measles virus (MeV) is a member of the family Paramixoviridae that causes a highly contagious respiratory disease but has emerged as a promising oncolytic platform. Previous studies of MeV entry focused on the identification of cellular receptors. However, the endocytic and trafficking pathways utilized during MeV entry remain poorly described. The contribution of each endocytic pathway has been examined in cells that express the MeV receptors SLAM (signaling lymphocyte-activating molecule) and PVRL4 (poliovirus receptor-like 4) (nectin-4). Recombinant MeVs expressing either firefly luciferase or green fluorescent protein together with a variety of inhibitors were used. The results showed that MeV uptake was dynamin independent in the Vero.hPVRL4, Vero.hSLAM, and PVRL4-positive MCF7 breast cancer cell lines. However, MeV infection was blocked by 5-(N-ethyl-N-propyl)amiloride (EIPA), the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. By using phalloidin staining, MeV entry was shown to induce actin rearrangements and the formation of membrane ruffles accompanied by transient elevated fluid uptake. Small interfering RNA (siRNA) knockdown of p21-activated kinase 1 (PAK1) demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner using a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer cells relied upon Rac1 and its effector PAK1 through a PVRL4-mediated macropinocytosis pathway. MeV entry into DLD-1 colon and HTB-20 breast cancer cells also appeared to use the same pathway. Overall, these findings provide new insight into the life cycle of MeV, which could lead to therapies that block virus entry or methods that improve the uptake of MeV by cancer cells during oncolytic therapy. IMPORTANCE In the past decades, measles virus (MeV) has emerged as a promising oncolytic platform. Previous studies concerning MeV entry focused mainly on the identification

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

PubMed

Ellipilli, Satheesh; Ganesh, Krishna N

2015-09-18

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

PubMed Central

Mercer, Jacob L.; Argus, Joseph P.; Crabtree, Donna M.; Keenan, Melissa M.; Wilks, Moses Q.; Chi, Jen-Tsan Ashley; Bensinger, Steven J.

2015-01-01

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease. PMID:26075887

C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway.

PubMed

Reich, Daniel P; Tyc, Katarzyna M; Bass, Brenda L

2018-02-01

Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of Caenorhabditis elegans development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in adr-1;adr-2 mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in adr-1;adr-2 embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway ( rrf-3 or ergo-1 ) in adr-1;adr-2 mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A) + RNA-seq revealed EAG down-regulation and antiviral gene induction in adr-1;adr-2;rrf-3 embryos, and these expression changes were dependent on rde-1 and rde-4 Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs. © 2018 Reich et al.; Published by Cold Spring Harbor Laboratory Press.

[Effects of sub-micro emulsion composition on cellular disposition of incorporated lipophilic drug].

PubMed

Sun, Xiao-Yi; Xiang, Zhi-Qiang; Wu, Shuo; Lv, Yuan-Yuan; Liang, Wen-Quan

2013-09-01

To investigate the effects of sub-micro emulsion composition on cellular uptake and disposition of incorporated lipophilic drug. Sub-micro emulsions containing 10 % oil, 1.2 % lecithin and 2.25 % glycerol were prepared, and the fluorescent agent coumarin 6 was used as a model drug. The effects of oil types, co-surfactants and cationic lipid on uptake and elimination kinetics of 6-coumarin in HeLa cells were studied. The uptake mechanism of sub-micro emulsions was further investigated. Oil type and Tweens had no influence on the cellular uptake. Modifications of surfactants with Span series increased the cellular influx, among which Span 20 with hydrophilic-lipophilic balance (HLB) value of 8.6 was the best enhancer. The intracellular drug level reached up to (46.09 ± 1.98)ng/μg protein which had significant difference with control group [(38.54 ± 0.34)ng/μg protein]. The positively charged emulsions significantly increased the uptake rate constant and elimination rate constant which were 4 times and 1.5 times of those in anionic groups, respectively. The uptake enhancement was also observed in cationic emulsions, cellular concentrations at plateau were (42.73 ± 0.84)ng/μg protein, which was about 3 times of that in anionic emulsions [(15.71 ± 0.74)ng/μg protein], when extracellular drug concentration kept at 100 ng/ml. Cationic emulsions delivered the payload mainly by direct drug transfer to contacted cells, while the negative ones depended on both drug passive diffusion and clathrin-mediated endocytosis of drug containing oil droplets which accounted for 20% of the intracellular drug. Interfacial characteristic of sub-micro emulsions such as co-surfactants HLB as well as zeta potentials can influence lipophilic drug both in cellular uptake and elimination.

Enhanced cellular uptake of LHRH-conjugated PEG-coated magnetite nanoparticles for specific targeting of triple negative breast cancer cells.

PubMed

Hu, J; Obayemi, J D; Malatesta, K; Košmrlj, A; Soboyejo, W O

2018-07-01

Targeted therapy is an emerging technique in cancer detection and treatment. This paper presents the results of a combined experimental and theoretical study of the specific targeting and entry of luteinizing hormone releasing hormone (LHRH)-conjugated PEG-coated magnetite nanoparticles into triple negative breast cancer (TNBC) cells and normal breast cells. The conjugated nanoparticles structures, cellular uptake of PEG-coated magnetite nanoparticles (MNPs) and LHRH-conjugated PEG-coated magnetite nanoparticles (LHRH-MNPs) into breast cancer cells and normal breast cells were investigated using a combination of transmission electron microscope, optical and confocal fluorescence microscopy techniques. The results show that the presence of LHRH enhances the uptake of LHRH-MNPs into TNBC cells. Nanoparticle entry into breast cancer cells is also studied using a combination of thermodynamics and kinetics models. The trends in the predicted nanoparticle entry times (into TNBC cells) and the size ranges of the engulfed nanoparticles (within the TNBC cells) are shown to be consistent with experimental observations. The implications of the results are then discussed for the specific targeting of TNBCs with LHRH-conjugated PEG-coated magnetite nanoparticles for the early detection and treatment of TNBC. Copyright © 2018. Published by Elsevier B.V.

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells

PubMed Central

Ma, Danjun; Wang, Jiarui; Zhao, Yingchun; Lee, Wai-Nang Paul; Xiao, Jing; Go, Vay Liang W.; Wang, Qi; Recker, Robert; Xiao, Gary Guishan

2011-01-01

Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (25 μM, 50 μM and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC method (mSILAC). Results A total of twenty-two protein spots and four phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of MAPK/ERK and TNF-α/NF-κB. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through one pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown and post-translational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. PMID:22158071

Design of Curcumin Loaded PLGA Nanoparticles Formulation with Enhanced Cellular Uptake, and Increased Bioactivity in vitro and Superior Bioavailability in vivo

PubMed Central

Anand, Preeta; Nair, Harish B.; Sung, Bokyung; Kunnumakkara, Ajaikumar B.; Yadav, Vivek R.; Tekmal, Rajeshwar R.; Aggarwal, Bharat B.

2011-01-01

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with antioxidant, anti-inflammatory, anti-proliferative, anticancer, antidiabetic, antirheumatic, and antiviral effects, but its optimum potential is limited by its lack of solubility in aqueous solvents and poor oral bioavailability. We employed a polymer-based nanoparticle approach to improve bioavailability. Curcumin was encapsulated with 97.5% efficiency in biodegradable nanoparticulate formulation based on poly (lactide-co-glycolide) (PLGA) and a stabilizer polyethylene glycol (PEG)-5000. Dynamic laser light scattering and transmission electron microscopy indicated a particle diameter of 80.9 nm. This curcumin, renamed from hereon “as curcumin (NP)”, was characterized for its biological activity. In vitro curcumin (NP) exhibited very rapid (2 h vs > 72 h) and more efficient cellular uptake then curcumin. Estrase staining revealed that curcumin (NP) was at least as potent as or more potent than curcumin in inducing apoptosis of leukemic cells and in suppressing proliferation of various tumor cell lines. When examined by electrophoretic gel shift mobility assay, curcumin (NP) was more active than curcumin in inhibiting TNF-induced NF-κB activation and in suppression of NF-κB-regulated proteins involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). In mice, curcumin (NP) was more bioavailable and had a longer half-life than curcumin. Overall we demonstrate that curcumin-loaded PLGA nanoparticles formulation has enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo over curcumin. PMID:19735646

Molecular and cellular characterisation of the zinc uptake (Znu) system of Nostoc punctiforme.

PubMed

Hudek, Lee; Pearson, Leanne A; Michalczyk, Agnes; Neilan, Brett A; Ackland, M Leigh

2013-11-01

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn(2+)-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn(2+)-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08(-), znuA18(-), znuB(-) and znuC(-) mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn(2+)-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA(-) mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur(-) mutant. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

IL-15 Activates the Jak3/STAT3 Signaling Pathway to Mediate Glucose Uptake in Skeletal Muscle Cells

PubMed Central

Krolopp, James E.; Thornton, Shantaé M.; Abbott, Marcia J.

2016-01-01

Myokines are specialized cytokines that are secreted from skeletal muscle (SKM) in response to metabolic stimuli, such as exercise. Interleukin-15 (IL-15) is a myokine with potential to reduce obesity and increase lean mass through induction of metabolic processes. It has been previously shown that IL-15 acts to increase glucose uptake in SKM cells. However, the downstream signals orchestrating the link between IL-15 signaling and glucose uptake have not been fully explored. Here we employed the mouse SKM C2C12 cell line to examine potential downstream targets of IL-15-induced alterations in glucose uptake. Following differentiation, C2C12 cells were treated overnight with 100 ng/ml of IL-15. Activation of factors associated with glucose metabolism (Akt and AMPK) and known downstream targets of IL-15 (Jak1, Jak3, STAT3, and STAT5) were assessed with IL-15 stimulation. IL-15 stimulated glucose uptake and GLUT4 translocation to the plasma membrane. IL-15 treatment had no effect on phospho-Akt, phospho-Akt substrates, phospho-AMPK, phospho-Jak1, or phospho-STAT5. However, with IL-15, phospho-Jak3 and phospho-STAT3 levels were increased along with increased interaction of Jak3 and STAT3. Additionally, IL-15 induced a translocation of phospho-STAT3 from the cytoplasm to the nucleus. We have evidence that a mediator of glucose uptake, HIF1α, expression was dependent on IL-15 induced STAT3 activation. Finally, upon inhibition of STAT3 the positive effects of IL-15 on glucose uptake and GLUT4 translocation were abolished. Taken together, we provide evidence for a novel signaling pathway for IL-15 acting through Jak3/STAT3 to regulate glucose metabolism. PMID:28066259

Cellular metabolism in colorectal carcinogenesis: Influence of lifestyle, gut microbiome and metabolic pathways.

PubMed

Hagland, Hanne R; Søreide, Kjetil

2015-01-28

The interconnectivity between diet, gut microbiota and cell molecular responses is well known; however, only recently has technology allowed the identification of strains of microorganisms harbored in the gastrointestinal tract that may increase susceptibility to cancer. The colonic environment appears to play a role in the development of colon cancer, which is influenced by the human metabolic lifestyle and changes in the gut microbiome. Studying metabolic changes at the cellular level in cancer be useful for developing novel improved preventative measures, such as screening through metabolic breath-tests or treatment options that directly affect the metabolic pathways responsible for the carcinogenicity. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Lognormal Distribution of Cellular Uptake of Radioactivity: Statistical Analysis of α-Particle Track Autoradiography

PubMed Central

Neti, Prasad V.S.V.; Howell, Roger W.

2010-01-01

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log-normal (LN) distribution function (J Nucl Med. 2006;47:1049–1058) with the aid of autoradiography. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analysis of these earlier data. Methods The measured distributions of α-particle tracks per cell were subjected to statistical tests with Poisson, LN, and Poisson-lognormal (P-LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL of 210Po-citrate. When cells were exposed to 67 kBq/mL, the P-LN distribution function gave a better fit; however, the underlying activity distribution remained log-normal. Conclusion The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:18483086

Differences in the cellular uptake and intracellular itineraries of amyloid beta proteins 40 and 42: ramifications for the Alzheimer's drug discovery.

PubMed

Omtri, Rajesh S; Davidson, Michael W; Arumugam, Balasubramaniam; Poduslo, Joseph F; Kandimalla, Karunya K

2012-07-02

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD), neurofibrillary tangles and parenchymal amyloid plaques, are downstream reflections of neurodegeneration caused by the intraneuronal accumulation of amyloid-β proteins (Aβ), particularly Aβ42 and Aβ40. While the neurotoxicity of more amyloidogenic but less abundant Aβ42 is well documented, the effect of Aβ40 on neurons has been understudied. The Aβ40 expression in the presymptomatic AD brain is ten times greater than that of Aβ42. However, the Aβ40:42 ratio decreases with AD progression and coincides with increased amyloid plaque deposition in the brain. Hence, it is thought that Aβ40 protects neurons from the deleterious effects of Aβ42. The pathophysiological pathways involved in the neuronal uptake of Aβ40 or Aβ42 have not been clearly elucidated. Lack of such critical information obscures therapeutic targets and thwarts rational drug development strategies aimed at preventing neurodegeneration in AD. The current study has shown that fluorescein labeled Aβ42 (F-Aβ42) is internalized by neurons via dynamin dependent endocytosis and is sensitive to membrane cholesterol, whereas the neuronal uptake of F-Aβ40 is energy independent and nonendocytotic. Following their uptake, both F-Aβ40 and F-Aβ42 did not accumulate in early/recycling endosomes; F-Aβ42 but not F-Aβ40 accumulated in late endosomes and in the vesicles harboring caveolin-1. Furthermore, F-Aβ42 demonstrated robust accumulation in the lysosomes and damaged their integrity, whereas F-Aβ40 showed only a sparse lysosomal accumulation. Such regulated trafficking along distinct pathways suggests that Aβ40 and Aβ42 exercise differential effects on neurons. These differences must be carefully considered in the design of a pharmacological agent intended to block the neurodegeneration triggered by Aβ proteins.

Physiological and molecular evidence for Pi uptake via the symbiotic pathway in a reduced mycorrhizal colonization mutant in tomato associated with a compatible fungus.

PubMed

Poulsen, Katrine H; Nagy, Réka; Gao, Ling-Ling; Smith, Sally E; Bucher, Marcel; Smith, F Andrew; Jakobsen, Iver

2005-11-01

A Lycopersicon esculentum mutant (rmc) is resistant to colonization by most arbuscular mycorrhizal fungi (AMF), but one Glomus intraradices isolate (WFVAM 23) develops arbuscules and vesicles in the rmc cortex. It is unknown whether the symbiotic phosphate (Pi)-uptake pathway is operational in this interaction. Hyphal uptake of (32)Pi and expression of plant Pi transporter genes were investigated in the rmc mutant and its wild-type progenitor (76R) associated with three AMF. Hyphae transferred (32)Pi in all symbioses with 76R and in the rmc-G. intraradices WFVAM 23 symbiosis. The other AMF did not colonize rmc. The Pi transporter-encoding LePT1 and LePT2 were expressed constitutively or in P-starved roots, respectively. The mycorrhiza-inducible Pi transporters LePT3 and LePT4 were expressed only in plants with AMF colonization and symbiotic (32)Pi transfer. LePT3 and LePT4 transcripts were reliable markers for a functional mycorrhizal uptake pathway in rmc. Our novel approach to the physiology and molecular biology of P transport can be applied to other arbuscular-mycorrhizal symbioses, irrespective of the size of plant responses.

Pathway towards Programmable Wave Anisotropy in Cellular Metamaterials

NASA Astrophysics Data System (ADS)

Celli, Paolo; Zhang, Weiting; Gonella, Stefano

2018-01-01

In this work, we provide a proof-of-concept experimental demonstration of the wave-control capabilities of cellular metamaterials endowed with populations of tunable electromechanical resonators. Each independently tunable resonator comprises a piezoelectric patch and a resistor-inductor shunt, and its resonant frequency can be seamlessly reprogrammed without interfering with the cellular structure's default properties. We show that, by strategically placing the resonators in the lattice domain and by deliberately activating only selected subsets of them, chosen to conform to the directional features of the beamed wave response, it is possible to override the inherent wave anisotropy of the cellular medium. The outcome is the establishment of tunable spatial patterns of energy distillation resulting in a nonsymmetric correction of the wave fields.

Reconciling the Krogh and Ussing interpretations of epithelial chloride transport - presenting a novel hypothesis for the physiological significance of the passive cellular chloride uptake.

PubMed

Larsen, Erik Hviid

2011-07-01

In 1937, August Krogh discovered a powerful active Cl(-) uptake mechanism in frog skin. After WWII, Hans Ussing continued the studies on the isolated skin and discovered the passive nature of the chloride uptake. The review concludes that the two modes of transport are associated with a minority cell type denoted as the γ-type mitochondria-rich (MR) cell, which is highly specialized for epithelial Cl(-) uptake whether the frog is in the pond of low [NaCl] or the skin is isolated and studied by Ussing chamber technique. One type of apical Cl(-) channels of the γ-MR cell is activated by binding of Cl(-) to an external binding site and by membrane depolarization. This results in a tight coupling of the uptake of Na(+) by principal cells and Cl(-) by MR cells. Another type of Cl(-) channels (probably CFTR) is involved in isotonic fluid uptake. It is suggested that the Cl(-) channels serve passive uptake of Cl(-) from the thin epidermal film of fluid produced by mucosal glands. The hypothesis is evaluated by discussing the turnover of water and ions of the epidermal surface fluid under terrestrial conditions. The apical Cl(-) channels close when the electrodiffusion force is outwardly directed as it is when the animal is in the pond. With the passive fluxes eliminated, the Cl(-) flux is governed by active transport and evidence is discussed that this is brought about by an exchange of cellular HCO(3) (-) with Cl(-) of the outside bath driven by an apical H(+) V-ATPase. © 2011 The Author. Acta Physiologica © 2011 Scandinavian Physiological Society.

Proteomic Analysis Implicates Dominant Alterations of RNA Metabolism and the Proteasome Pathway in the Cellular Response to Carbon-Ion Irradiation

PubMed Central

Xie, Da-Fei; Xie, Yi; Liu, Xiao-Dan; Wang, Qi; Sui, Li; Song, Man; Zhang, Hong; Zhou, Jianhua; Zhou, Ping-Kun

2016-01-01

Radiotherapy with heavy ions is considered advantageous compared to irradiation with photons due to the characteristics of the Braggs peak and the high linear energy transfer (LET) value. To understand the mechanisms of cellular responses to different LET values and dosages of heavy ion radiation, we analyzed the proteomic profiles of mouse embryo fibroblast MEF cells exposed to two doses from different LET values of heavy ion 12C. Total proteins were extracted from these cells and examined by Q Exactive with Liquid Chromatography (LC)—Electrospray Ionization (ESI) Tandem MS (MS/MS). Using bioinformatics approaches, differentially expressed proteins with 1.5 or 2.0-fold changes between different dosages of exposure were compared. With the higher the dosage and/or LET of ion irradiation, the worse response the cells were in terms of protein expression. For instance, compared to the control (0 Gy), 771 (20.2%) proteins in cells irradiated at 0.2 Gy of carbon-ion radiation with 12.6 keV/μm, 313 proteins (8.2%) in cells irradiated at 2 Gy of carbon-ion radiation with 12.6 keV/μm, and 243 proteins (6.4%) in cells irradiated at 2 Gy of carbon-ion radiation with 31.5 keV/μm exhibited changes of 1.5-fold or greater. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Munich Information Center for Protein Sequences (MIPS) analysis, and BioCarta analysis all indicated that RNA metabolic processes (RNA splicing, destabilization and deadenylation) and proteasome pathways may play key roles in the cellular response to heavy-ion irradiation. Proteasome pathways ranked highest among all biological processes associated with heavy carbon-ion irradiation. In addition, network analysis revealed that cellular pathways involving proteins such as Col1a1 and Fn1 continued to respond to high dosages of heavy-ion irradiation, suggesting that these pathways still protect cells against damage. However, pathways such as those involving Ikbkg1 responded

Role of cellular communication in the pathways of radiation-induced biological damage

NASA Astrophysics Data System (ADS)

Ballarini, Francesca; Facoetti, Angelica; Mariotti, Luca; Nano, Rosanna; Ottolenghi, Andrea

During the last decade, a large number of experimental studies on the so-called "non-targeted effects", in particular bystander effects, outlined that cellular communication plays a signifi- cant role in the pathways leading to radiation-induced biological damage. This might imply a paradigm shift in (low-dose) radiobiology, according to which one has to consider the response of groups of cells behaving like a population rather than single cells behaving as individuals. Furthermore, bystander effects, which are observed both for lethal endpoints (e.g. clonogenic inactivation and apoptosis) and for non-lethal ones (e.g. mutations and neoplastic transformation), tend to show non-linear dose responses characterized by a sharp increase followed by a plateau. This might have significant consequences in terms of low-dose risk, which is generally calculated on the basis of the "Linear No Threshold" hypothesis. Although it is known that two types of cellular communication (i.e. via gap junctions and/or molecular messengers diffusing in the extra-cellular environment, such as cytokines) play a major role, it is of utmost importance to better understand the underlying mechanisms, and how such mechanisms can be modulated by ionizing radiation. Though the "final" goal is to elucidate the in vivo scenario, in the meanwhile also in vitro studies can provide useful insights. In the present paper we will discuss key issues on the mechanisms underlying non-targeted effects and, more generally, cell communication, with focus on candidate molecular signals. Theoretical models and simulation codes can be of help in elucidating such mechanisms. In this framework, we will present a model and Monte Carlo code, under development at the University of Pavia, simulating the release, diffusion and internalization of candidate signals (typically cytokines) travelling in the extra-cellular environment, both by unirradiated (i.e., control) cells and by irradiated cells. The focus will be on the

L-Proline uptake in Saccharomyces cerevisiae mitochondria can contribute to bioenergetics during nutrient stress as alternative mitochondrial fuel.

PubMed

Pallotta, Maria Luigia

2014-01-01

L-Proline (pyrrolidine-2-carboxylic acid) is a distinctive metabolite both biochemically and biotechnologically and is currently recognized to have a cardinal role in gene expression and cellular signaling pathways in stress response. Proline-fueled mitochondrial metabolism involves the oxidative conversion of L-Proline to L-Glutamate in two enzymatic steps by means of Put1p and Put2p that help Saccharomyces cerevisiae to respond to changes in the nutritional environment by initiating the breakdown of L-Proline as a source for nitrogen, carbon, and energy. Compartmentalization of L-Proline catabolic pathway implies that extensive L-Proline transport must take place between the cytosol where its biogenesis via Pro1p, Pro2p, Pro3p occurs and mitochondria. L-Proline uptake in S. cerevisiae purified and active mitochondria was investigated by swelling experiments, oxygen uptake and fluorimetric measurement of a membrane potential generation (ΔΨ). Our results strongly suggest that L-Proline uptake occurs via a carried-mediated process as demonstrated by saturation kinetics and experiments with N-ethylmaleimide, a pharmacological compound that is a cysteine-modifying reagent in hydrophobic protein domains and that inhibited mitochondrial transport. Plasticity of S. cerevisiae cell biochemistry according to background fluctuations is an important factor of adaptation to stress. Thus L-Proline → Glutamate route feeds Krebs cycle providing energy and anaplerotic carbon for yeast survival.

Curcumin Encapsulated into Methoxy Poly(Ethylene Glycol) Poly(ε-Caprolactone) Nanoparticles Increases Cellular Uptake and Neuroprotective Effect in Glioma Cells.

PubMed

Marslin, Gregory; Sarmento, Bruno Filipe Carmelino Cardoso; Franklin, Gregory; Martins, José Alberto Ribeiro; Silva, Carlos Jorge Ribeiro; Gomes, Andreia Ferreira Castro; Sárria, Marisa Passos; Coutinho, Olga Maria Fernandes Pereira; Dias, Alberto Carlos Pires

2017-03-01

Curcumin is a natural polyphenolic compound isolated from turmeric ( Curcuma longa ) with well-demonstrated neuroprotective and anticancer activities. Although curcumin is safe even at high doses in humans, it exhibits poor bioavailability, mainly due to poor absorption, fast metabolism, and rapid systemic elimination. To overcome these issues, several approaches, such as nanoparticle-mediated targeted delivery, have been undertaken with different degrees of success. The present study was conducted to compare the neuroprotective effect of curcumin encapsulated in poly( ε -caprolactone) and methoxy poly(ethylene glycol) poly( ε -caprolactone) nanoparticles in U251 glioblastoma cells. Prepared nanoparticles were physically characterized by laser doppler anemometry, transmission electron microscopy, and X-ray diffraction. The results from laser doppler anemometry confirmed that the size of poly( ε -caprolactone) and poly(ethylene glycol) poly( ε -caprolactone) nanoparticles ranged between 200-240 nm for poly( ε -caprolactone) nanoparticles and 30-70 nm for poly(ethylene glycol) poly( ε -caprolactone) nanoparticles, and transmission electron microscopy images revealed their spherical shape. Treatment of U251 glioma cells and zebrafish embryos with poly( ε -caprolactone) and poly(ethylene glycol) poly( ε -caprolactone) nanoparticles loaded with curcumin revealed efficient cellular uptake. The cellular uptake of poly(ethylene glycol) poly( ε -caprolactone) nanoparticles was higher in comparison to poly( ε -caprolactone) nanoparticles. Moreover, poly(ethylene glycol) poly( ε -caprolactone) di-block copolymer-loaded curcumin nanoparticles were able to protect the glioma cells against tBHP induced-oxidative damage better than free curcumin. Together, our results show that curcumin-loaded poly(ethylene glycol) poly( ε -caprolactone) di-block copolymer nanoparticles possess significantly stronger neuroprotective effect in U251 human glioma cells compared to

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

PubMed Central

Pan, Jie; Liu, Wei-Jiao; Hua, Chao; Wang, Li-Li; Wan, Dong; Gong, Jun-Bo

2015-01-01

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection. PMID:26487962

Noscapinoids bearing silver nanocrystals augmented drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1, mouse melanoma skin cancer cells.

PubMed

Soni, Naina; Jyoti, Kiran; Jain, Upendra Kumar; Katyal, Anju; Chandra, Ramesh; Madan, Jitender

2017-06-01

Noscapine (Nos) and reduced brominated analogue of noscapine (Red-Br-Nos) prevent cellular proliferation and induce apoptosis in cancer cells either alone or in combination with other chemotherapeutic drugs. However, owing to poor physicochemical properties, Nos and Red-Br-Nos have demonstrated their anticancer activity at higher and multiple doses. Therefore, in present investigation, silver nanocrystals of noscapinoids (Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals) were customized to augment drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1 mouse melanoma cancer cells. Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals were prepared separately by precipitation method. The mean particle size of Nos-Ag 2+ nanocrystals was measured to be 25.33±3.52nm, insignificantly (P>0.05) different from 27.43±4.51nm of Red-Br-Nos-Ag 2+ nanocrystals. Furthermore, zeta-potential of Nos-Ag 2+ nanocrystals was determined to be -25.3±3.11mV significantly (P<0.05) different from -15.2±3.33mV of Red-Br-Nos-Ag 2+ nanocrystals. The shape of tailored nanocrystals was slightly spherical and or irregular in shape. The architecture of Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals was crystalline in nature. FT-IR spectroscopy evinced the successful interaction of Ag 2+ nanocrystals with Nos and Red-Br-Nos, respectively. The superior therapeutic efficacy of tailored nanocrystals was measured in terms of enhanced cytotoxicity, apoptosis and cellular uptake. The Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals exhibited an IC 50 of 16.6μM and 6.5μM, significantly (P<0.05) lower than 38.5μM of Nos and 10.3μM of Red-Br-Nos, respectively. Finally, cellular morphological alterations in B16F1 cells upon internalization of Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals provided the evidences for accumulation within membrane-bound cytoplasmic vacuoles and in enlarged lysosomes and thus triggered mitochondria mediated apoptosis via

Bupivacaine-induced cellular entry of QX-314 and its contribution to differential nerve block

PubMed Central

Brenneis, C; Kistner, K; Puopolo, M; Jo, S; Roberson, DP; Sisignano, M; Segal, D; Cobos, EJ; Wainger, BJ; Labocha, S; Ferreirós, N; Hehn, C; Tran, J; Geisslinger, G; Reeh, PW; Bean, BP; Woolf, C J

2014-01-01

Background and Purpose: Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. Experimental Approach: We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. Key Results: Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. Conclusions and Implications: Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful. PMID:24117225

Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain.

PubMed

Haug, Gerd; Wilde, Christian; Leemhuis, Jost; Meyer, Dieter K; Aktories, Klaus; Barth, Holger

2003-12-30

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

Targeting genes in insulin-associated signalling pathway, DNA damage, cell proliferation and cell differentiation pathways by tocotrienol-rich fraction in preventing cellular senescence of human diploid fibroblasts.

PubMed

Durani, L W; Jaafar, F; Tan, J K; Tajul Arifin, K; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

2015-01-01

Tocotrienols have been known for their antioxidant properties besides their roles in cellular signalling, gene expression, immune response and apoptosis. This study aimed to determine the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs) by targeting the genes in senescence-associated signalling pathways. Real time quantitative PCR (qRT-PCR) was utilized to evaluate the expression of genes involved in these pathways. Our findings showed that SOD1 and CCS-1 were significantly down-regulated in pre-senescent cells while CCS-1 and PRDX6 were up-regulated in senescent cells (p<0.05). Treatment with TRF significantly down-regulated SOD1 in pre-senescent and senescent HDFs, up-regulated SOD2 in senescent cells, CAT in young HDFs, GPX1 in young and pre-senescent HDFs, and CCS-1 in young, pre-senescent and senescent HDFs (p<0.05). TRF treatment also caused up-regulation of FOXO3A in all age groups of cells (p<0.05). The expression of TP53, PAK2 and CDKN2A was significantly increased in senescent HDFs and treatment with TRF significantly down-regulated TP53 in senescent cells (p<0.05). MAPK14 was significantly up-regulated (p<0.05) in senescent HDFs while no changes was observed on the expression of JUN. TRF treatment, however, down-regulated MAPK14 in young and senescent cells and up-regulated JUN in young and pre-senescent HDFs (p<0.05). TRF modulated the expression of genes involved in senescence-associated signalling pathways during replicative senescence of HDFs.

Preparation of large porous deslorelin-PLGA microparticles with reduced residual solvent and cellular uptake using a supercritical carbon dioxide process.

PubMed

Koushik, Kavitha; Kompella, Uday B

2004-03-01

The purpose of this study was to prepare large-porous peptide-encapsulating polymeric particles with low residual solvent that retain deslorelin integrity, sustain drug release, and exhibit reduced epithelial and macrophage uptake. We hypothesized that supercritical carbon dioxide (SC CO2) pressure-quench treatment of microparticles prepared using conventional approach expands these particles and extracts the residual organic solvent. Initial studies with crystalline L-lactide (L-PLA) and amorphous copolymers of lactide-co-glycolide (PLGA) 50:50, 65:35, and 75:25 indicated that PLGA 50:50 was the most amenable to morphological changes upon SC CO2 treatment. Therefore, we prepared deslorelin-PLGA (50:50) microparticles using the conventional emulsion-solvent evaporation method, and in a second step equilibrated with SC CO2 at various temperatures (33-37 degrees C) and pressures (1200-2000 psi) for discrete intervals followed by rapid isothermal depressurization. The particles were then characterized for morphology, polymer thermal properties, particle size, porosity, bulk density, and residual solvent content. Also, deslorelin integrity, conformation, release, and cellular uptake before and after SC CO2 treatment was determined. Upon SC CO2 treatment (1200 psi, 33 degrees C for 30 min), the mean particle size of the deslorelin PLGA microparticles increased from 2.2 to 13.8 microm, the mean porosity increased from 39 to 92.38% the mean pore diameter increased from 90 to 190 nm, the mean bulk density reduced from 0.7 to 0.082 g/cc, mass spectrometry indicated structural integrity of released deslorelin, the circular dichroism spectrum indicated stabilization of beta-turn conformation, and the scanning electron microscopy confirmed increased particle size and pore formation. The deslorelin release was sustained during the 7-day study period. Also, the peak Tg of PLGA decreased from 51 to 45 degrees C, and the residual solvent content was reduced from 4500 ppm to below

Cell uptake survey of pegylated nanographene oxide.

PubMed

Vila, M; Portolés, M T; Marques, P A A P; Feito, M J; Matesanz, M C; Ramírez-Santillán, C; Gonçalves, G; Cruz, S M A; Nieto, A; Vallet-Regi, M

2012-11-23

Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml(-1) pegylated GO solutions. GO uptake kinetics revealed differences in the agent's uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy.

Cellular Uptake and Tissue Biodistribution of Functionalized Gold Nanoparticles and Nanoclusters.

PubMed

Escudero-Francos, María A; Cepas, Vanesa; González-Menédez, Pedro; Badía-Laíño, Rosana; Díaz-García, Marta E; Sainz, Rosa M; Mayo, Juan C; Hevia, David

2017-02-01

In this study, the in vitro uptake by fibroblasts and in vivo biodistribution of 15 nm 11-mercaptoundecanoicacid-protected gold nanoparticles (AuNPs-MUA) and 3 nm glutathione- and 3 nm bovine serum albumin-protected gold nanoclusters (AuNCs@GSH and AuNCs@BSA, respectively) were evaluated. In vitro cell viability was examined after gold nanoparticle treatment for 48 h, based on MTT assays and analyses of morphological structure, the cycle cell, cellular doubling time, and the gold concentration in cells. No potential toxicity was observed at any studied concentration (up to 10 ppm) for AuNCs@GSH and AuNCs@BSA, whereas lower cell viability was observed for AuNPs-MUA at 10 ppm than for other treatments. Neither morphological damage nor modifications to the cell cycle and doubling time were detected after contact with nanoparticles. Associations between cells and AuNPs and AuNCs were demonstrated by inductively coupled plasma mass spectrometry (ICP-MS). AuNCs@GSH exhibited fluorescence emission at 611 nm, whereas AuNCs@BSA showed a band at 640 nm. These properties were employed to confirm their associations with cells by fluorescence confocal microscopy; both clusters were observed in cells and maintained their original fluorescence. In vivo assays were performed using 9 male mice treated with 1.70 μg Au/g body weight gold nanoparticles for 24 h. ICP-MS measurements showed a different biodistribution for each type of nanoparticle; AuNPs-MUA mainly accumulated in the brain, AuNCs@GSH in the kidney, and AuNCs@BSA in the liver and spleen. Spleen indexes were not affected by nanoparticle treatment; however, AuNCs@BSA increased the thymus index significantly from 1.28 to 1.79, indicating an immune response. These nanoparticles have great potential as organ-specific drug carriers and for diagnosis, photothermal therapy, and imaging.

Log Normal Distribution of Cellular Uptake of Radioactivity: Statistical Analysis of Alpha Particle Track Autoradiography

PubMed Central

Neti, Prasad V.S.V.; Howell, Roger W.

2008-01-01

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a log normal distribution function (J Nucl Med 47, 6 (2006) 1049-1058) with the aid of an autoradiographic approach. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analyses of these data. Methods The measured distributions of alpha particle tracks per cell were subjected to statistical tests with Poisson (P), log normal (LN), and Poisson – log normal (P – LN) models. Results The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL 210Po-citrate. When cells were exposed to 67 kBq/mL, the P – LN distribution function gave a better fit, however, the underlying activity distribution remained log normal. Conclusions The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution. PMID:16741316

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levesque, Martine; Martineau, Corine; Jumarie, Catherine

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependentmore » manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of {sup 109}Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd{sup 2+} species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive

Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA

PubMed Central

Prevette, Lisa E.; Mullen, Douglas G.; Banaszak Holl, Mark M.

2010-01-01

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression. PMID:20349965

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

PubMed Central

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-01-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

NASA Astrophysics Data System (ADS)

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-01

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

PubMed

Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

2015-06-12

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

PubMed Central

Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

2012-01-01

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition.

PubMed

Sheokand, Navdeep; Kumar, Santosh; Malhotra, Himanshu; Tillu, Vikas; Raje, Chaaya Iyengar; Raje, Manoj

2013-06-01

The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron. These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays. This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized. Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis. Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

Dietary uptake of Cu sorbed to hydrous iron oxide is linked to cellular toxicity and feeding inhibition in a benthic grazer

USGS Publications Warehouse

Cain, Daniel J.; Croteau, Marie-Noele; Fuller, Christopher C.; Ringwood, Amy H.

2016-01-01

Whereas feeding inhibition caused by exposure to contaminants has been extensively documented, the underlying mechanism(s) are less well understood. For this study, the behavior of several key feeding processes, including ingestion rate and assimilation efficiency, that affect the dietary uptake of Cu were evaluated in the benthic grazer Lymnaea stagnalis following 4–5 h exposures to Cu adsorbed to synthetic hydrous ferric oxide (Cu–HFO). The particles were mixed with a cultured alga to create algal mats with Cu exposures spanning nearly 3 orders of magnitude at variable or constant Fe concentrations, thereby allowing first order and interactive effects of Cu and Fe to be evaluated. Results showed that Cu influx rates and ingestion rates decreased as Cu exposures of the algal mat mixture exceeded 104 nmol/g. Ingestion rate appeared to exert primary control on the Cu influx rate. Lysosomal destabilization rates increased directly with Cu influx rates. At the highest Cu exposure where the incidence of lysosomal membrane damage was greatest (51%), the ingestion rate was suppressed 80%. The findings suggested that feeding inhibition was a stress response emanating from excessive uptake of dietary Cu and cellular toxicity.

Cellular uptake and anticancer activity of carboxylated gallium corroles.

PubMed

Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H; Gray, Harry B; Termini, John; Lim, Punnajit

2016-04-19

We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax= 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 > 3 > 2 > 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging.

Cellular uptake and anticancer activity of carboxylated gallium corroles

PubMed Central

Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H.; Gray, Harry B.; Termini, John; Lim, Punnajit

2016-01-01

We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50 values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50 values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax = 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 >> 3 > 2 >> 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging. PMID:27044076

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication

PubMed Central

Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J. W.; Pankiewicz, Krzysztof W.; Sprengers, Dave; Raat, Nicolaas J. H.; Metselaar, Herold J.; Peppelenbosch, Maikel P.

2016-01-01

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway

Mechanisms of cell uptake, inflammatory potential and protein corona effects with gold nanoparticles.

PubMed

Li, Yang; Monteiro-Riviere, Nancy A

2016-12-01

To assess inflammation, cellular uptake and endocytic mechanisms of gold nanoparticles (AuNP) in human epidermal keratinocytes with and without a protein corona. Human epidermal keratinocytes were exposed to 40 and 80 nm AuNP with lipoic acid, polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) coatings with and without a protein corona up to 48 h. Inhibitors were selected to characterize endocytosis. BPEI-AuNP showed the greatest uptake, while PEG-AuNP had the least. Protein coronas decreased uptake and affected their mechanism. AuNP uptake was energy-dependent, except for 40 nm lipoic-AuNP. Most AuNP were internalized by clathrin and lipid raft-mediated endocytosis, except for 40 nm PEG was by raft/noncaveolae mediated endocytosis. Coronas inhibited caveolae-mediated-endocytosis with lipoic acid and BPEI-AuNP and altered 40 nm PEG-AuNP from raft/noncaveolae to clathrin. Inflammatory responses decreased with a plasma corona. Results suggest protein coronas significantly affect cellular uptake and inflammatory responses of AuNP.

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

PubMed

Poulin, Patrick; Burczynski, Frank J; Haddad, Sami

2016-02-01

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations

Effect of Molecular Structure of Cationic Surfactants on Biophysical Interactions of the Surfactant-modified Nanoparticles with a Model Membrane and Cellular Uptake

PubMed Central

Peetla, Chiranjeevi; Labhasetwar, Vinod

2009-01-01

The aim of this study was to test the hypothesis that the molecular structure of cationic surfactants at the nanoparticle (NP)-interface influences the biophysical interactions of NPs with a model membrane and cellular uptake of NPs. Polystyrene NPs (surfactant free, 130 nm) were modified with cationic surfactants. These surfactants were of either dichained (didodecyldimethylammonium bromide [DMAB]) or single chained (cetyltrimethylammonium bromide [CTAB] and dodecyltrimethylammonium bromide [DTAB]) forms, the latter two with different hydrophobic chain lengths. Biophysical interactions of these surfactant-modified NPs with an endothelial cell model membrane (EMM) were studied using a Langmuir film balance. Changes in surface pressure (SP) of EMM as a function of time following interaction with NPs and in the compression isotherm (π - A) of the lipid mixture of EMM in the presence of NPs were analyzed. Langmuir-Schaeffer (LS) films, which are EMMs that have been transferred onto a suitable substrate, were imaged by atomic force microscopy (AFM), and the images were analyzed to determine the mechanisms of the NP-EMM interaction. DMAB-modified NPs showed a greater increase in SP and a shift towards higher mean molecular area (mmA) than CTAB- and DTAB-modified NPs, indicating stronger interactions of DMAB-modified NPs with the EMM. However, analysis of the AFM phase and height images of the LS films revealed that both DMAB- and CTAB-modified NPs interacted with the EMM but via different mechanisms: DMAB-modified NPs penetrated the EMM, thus explaining the increase in SP, whereas CTAB-modified NPs anchored onto the EMM's condensed lipid domains, and hence did not cause any significant change in SP. Human umbilical vein endothelial cells showed greater uptake of DMAB- and CTAB-modified NPs than of DTAB-modified or unmodified NPs. We conclude that (i) the dichained and single-chained cationic surfactants on NPs have different mechanisms of interaction with the model

PFOS induces adipogenesis and glucose uptake in association with activation of Nrf2 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Jialin; Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881; Shimpi, Prajakta

PFOS is a chemical of nearly ubiquitous exposure in humans. Recent studies have associated PFOS exposure to adipose tissue-related effects. The present study was to determine whether PFOS alters the process of adipogenesis and regulates insulin-stimulated glucose uptake in mouse and human preadipocytes. In murine-derived 3T3-L1 preadipocytes, PFOS enhanced hormone-induced differentiation to adipocytes and adipogenic gene expression, increased insulin-stimulated glucose uptake at concentrations ranging from 10 to 100 μM, and enhanced Glucose transporter type 4 and Insulin receptor substrate-1 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NAD(P)H dehydrogenase, quinone 1 and Glutamate-cysteine ligase, catalytic subunit were significantly induced in 3T3-L1more » cells treated with PFOS, along with a robust induction of Antioxidant Response Element (ARE) reporter in mouse embryonic fibroblasts isolated from ARE-hPAP transgenic mice by PFOS treatment. Chromatin immunoprecipitation assays further illustrated that PFOS increased Nrf2 binding to ARE sites in mouse Nqo1 promoter, suggesting that PFOS activated Nrf2 signaling in murine-derived preadipocytes. Additionally, PFOS administration in mice (100 μg/kg/day) induced adipogenic gene expression and activated Nrf2 signaling in epididymal white adipose tissue. Moreover, the treatment on human visceral preadipocytes illustrated that PFOS (5 and 50 μM) promoted adipogenesis and increased cellular lipid accumulation. It was observed that PFOS increased Nrf2 binding to ARE sites in association with Nrf2 signaling activation, induction of Peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α expression, and increased adipogenesis. This study points to a potential role of PFOS in dysregulation of adipose tissue expandability, and warrants further investigations on the adverse effects of persistent pollutants on human health. - Highlights: • PFOS induces adipogenesis in

Adverse Outcome Pathway (AOP) Network Development for ...

EPA Pesticide Factsheets

Adverse outcome pathways (AOPs) are descriptive biological sequences that start from a molecular initiating event (MIE) and end with an adverse health outcome. AOPs provide biological context for high throughput chemical testing and further prioritize environmental health risk research. According to the Organization for Economic Co-operation and Development guidelines, AOPs are pathways with one MIE anchored to an adverse outcome (AO) by key events (KEs) and key event relationships (KERs). However, this approach does not always capture the cumulative impacts of multiple MIEs on the AO. For example, hepatic lipid flux due to chemical-induced toxicity initiates from multiple ligand-activated receptors and signaling pathways that cascade across biology to converge upon a common fatty liver (FL, also known as steatosis) outcome. To capture this complexity, a top-down strategy was used to develop a FL AOP network (AOPnet). Literature was queried based on the terms steatosis, fatty liver, cirrhosis, and hepatocellular carcinoma. Search results were analyzed for physiological and pathophysiological organ level, cellular and molecular processes, as well as pathway intermediates, to identify potential KEs and MIEs that are key for hepatic lipid metabolism, maintenance, and dysregulation. The analysis identified four apical KE nodes (hepatic fatty acid uptake, de novo fatty acid and lipid synthesis, fatty acid oxidation, and lipid efflux) juxtaposed to the FL AO. The apic

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

Potential Mechanisms of Action of Dietary Phytochemicals for Cancer Prevention by Targeting Cellular Signaling Transduction Pathways.

PubMed

Chen, Hongyu; Liu, Rui Hai

2018-04-04

Cancer is a severe health problem that significantly undermines life span and quality. Dietary approach helps provide preventive, nontoxic, and economical strategies against cancer. Increased intake of fruits, vegetables, and whole grains are linked to reduced risk of cancer and other chronic diseases. The anticancer activities of plant-based foods are related to the actions of phytochemicals. One potential mechanism of action of anticancer phytochemicals is that they regulate cellular signal transduction pathways and hence affects cancer cell behaviors such as proliferation, apoptosis, and invasion. Recent publications have reported phytochemicals to have anticancer activities through targeting a wide variety of cell signaling pathways at different levels, such as transcriptional or post-transcriptional regulation, protein activation and intercellular messaging. In this review, we discuss major groups of phytochemicals and their regulation on cell signaling transduction against carcinogenesis via key participators, such as Nrf2, CYP450, MAPK, Akt, JAK/STAT, Wnt/β-catenin, p53, NF-κB, and cancer-related miRNAs.

A Chemical Biology Approach to Interrogate Quorum Sensing Regulated Behaviors at the Molecular and Cellular Level

PubMed Central

Lowery, Colin A.; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan A.; Mee, Jenny M.; Cravatt, Benjamin F.; Miller, Samuel I.; Kaufmann, Gunnar F.; Janda, Kim D.

2013-01-01

SUMMARY Small molecule probes have been employed extensively to explore biological systems and elucidate cellular signaling pathways. In this study, we utilize an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering new processes regulated by AI-2-based quorum sensing (QS), a mechanism of bacterial intracellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intracellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. PMID:23890008

A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

PubMed

Lowery, Colin A; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan; Lively, Jenny M; Cravatt, Benjamin F; Miller, Samuel I; Kaufmann, Gunnar F; Janda, Kim D

2013-07-25

Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cellular and molecular pathways of extremely-low-frequency electromagnetic field interactions with living systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tenforde, T.S.

1992-06-01

There is growing evidence that environmental electric and magnetic fields in the extremely-low-frequency (ELF) band below 300 Hz can influence biological functions by mechanisms that are only poorly understood at the present time. The primary objectives of this paper are to review the physical properties of ELF fields, their interactions with living systems at the tissue, cellular, and subcellular levels, and the key role of cell membranes ;in the transduction of signals from imposed ELF fields. Topics of discussion include signal-to-noise ratios for single cells and cell aggregates, resonance phenomena involving a combination of static and ELF magnetic fields, andmore » the possible influence of ELF fields on molecular signaling pathways that involve membrane receptors and cytoplasmic second messengers.« less

Luminescent single-walled carbon nanotube-sensitized europium nanoprobes for cellular imaging

PubMed Central

Avti, Pramod K; Sitharaman, Balaji

2012-01-01

Lanthanoid-based optical probes with excitation wavelengths in the ultra-violet (UV) range (300–325 nm) have been widely developed as imaging probes. Efficient cellular imaging requires that lanthanoid optical probes be excited at visible wavelengths, to avoid UV damage to cells. The efficacy of europium-catalyzed single-walled carbon nanotubes (Eu-SWCNTs), as visible nanoprobes for cellular imaging, is reported in this study. Confocal fluorescence microscopy images of breast cancer cells (SK-BR-3 and MCF-7) and normal cells (NIH 3T3), treated with Eu-SWCNT at 0.2 μg/mL concentration, showed bright red luminescence after excitation at 365 nm and 458 nm wavelengths. Cell viability analysis showed no cytotoxic effects after the incubation of cells with Eu-SWCNTs at this concentration. Eu-SWCNT uptake is via the endocytosis mechanism. Labeling efficiency, defined as the percentage of incubated cells that uptake Eu-SWCNT, was 95%–100% for all cell types. The average cellular uptake concentration was 6.68 ng Eu per cell. Intracellular localization was further corroborated by transmission electron microscopy and Raman microscopy. The results indicate that Eu-SWCNT shows potential as a novel cellular imaging probe, wherein SWCNT sensitizes Eu3+ ions to allow excitation at visible wavelengths, and stable time-resolved red emission. The ability to functionalize biomolecules on the exterior surface of Eu-SWCNT makes it an excellent candidate for targeted cellular imaging. PMID:22619533

Interplay of drug metabolizing enzymes with cellular transporters.

PubMed

Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

2014-11-01

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Evaluation of in-vitro cytotoxicity and cellular uptake efficiency of zidovudine-loaded solid lipid nanoparticles modified with Aloe Vera in glioma cells.

PubMed

K S, Joshy; Sharma, Chandra P; Kalarikkal, Nandakumar; Sandeep, K; Thomas, Sabu; Pothen, Laly A

2016-09-01

Zidovudine loaded solid lipid nanoparticles of stearic acid modified with Aloe Vera (AV) have been prepared via simple emulsion solvent evaporation method which showed excellent stability at room temperature and refrigerated condition. The nanoparticles were examined by Fourier transform infrared spectroscopy (FT-IR), which revealed the overlap of the AV absorption peak with the absorption peak of modified stearic acid nanoparticles. The inclusion of AV to stearic acid decreased the crystallinity and improved the hydrophilicity of lipid nanoparticles and thereby improved the drug loading efficacy of lipid nanoparticles. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging revealed that, the average particle size of unmodified (bare) nanoparticles was 45.66±12.22nm and modified solid lipid nanoparticles showed an average size of 265.61±80.44nm. Solid lipid nanoparticles with well-defined morphology were tested in vitro for their possible application in drug delivery. Cell culture studies using C6 glioma cells on the nanoparticles showed enhanced growth and proliferation of cells without exhibiting any toxicity. In addition, normal cell morphology and improved uptake were observed by fluorescence microscopy images of rhodamine labeled modified solid lipid nanoparticles compared with unmodified nanoparticles. The cellular uptake study suggested that these nanoparticles could be a promising drug delivery system to enhance the uptake of antiviral drug by brain cells and it could be a suitable drug carrier system for the treatment of HIV. Copyright © 2016 Elsevier B.V. All rights reserved.

Cellular uptake: lessons from supramolecular organic chemistry.

PubMed

Gasparini, Giulio; Bang, Eun-Kyoung; Montenegro, Javier; Matile, Stefan

2015-07-04

The objective of this Feature Article is to reflect on the importance of established and emerging principles of supramolecular organic chemistry to address one of the most persistent problems in life sciences. The main topic is dynamic covalent chemistry on cell surfaces, particularly disulfide exchange for thiol-mediated uptake. Examples of boronate and hydrazone exchange are added for contrast, comparison and completion. Of equal importance are the discussions of proximity effects in polyions and counterion hopping, and more recent highlights on ring tension and ion pair-π interactions. These lessons from supramolecular organic chemistry apply to cell-penetrating peptides, particularly the origin of "arginine magic" and the "pyrenebutyrate trick," and the currently emerging complementary "disulfide magic" with cell-penetrating poly(disulfide)s. They further extend to the voltage gating of neuronal potassium channels, gene transfection, and the delivery of siRNA. The collected examples illustrate that the input from conceptually innovative chemistry is essential to address the true challenges in biology beyond incremental progress and random screening.

Lysine-functionalized nanodiamonds as gene carriers: development of stable colloidal dispersion for in vitro cellular uptake studies and siRNA delivery application

PubMed Central

Alwani, Saniya; Kaur, Randeep; Michel, Deborah; Chitanda, Jackson M; Verrall, Ronald E; Karunakaran, Chithra; Badea, Ildiko

2016-01-01

Purpose Nanodiamonds (NDs) are emerging as an attractive tool for gene therapeutics. To reach their full potential for biological application, NDs should maintain their colloidal stability in biological milieu. This study describes the behavior of lysine-functionalized ND (lys-ND) in various dispersion media, with an aim to limit aggregation and improve the colloidal stability of ND-gene complexes called diamoplexes. Furthermore, cellular and macromolecular interactions of lys-NDs are also analyzed in vitro to establish the understanding of ND-mediated gene transfer in cells. Methods lys-NDs were synthesized earlier through covalent conjugation of lysine amino acid to carboxylated NDs surface generated through re-oxidation in strong oxidizing acids. In this study, dispersions of lys-NDs were prepared in various media, and the degree of sedimentation was monitored for 72 hours. Particle size distributions and zeta potential measurements were performed for a period of 25 days to characterize the physicochemical stability of lys-NDs in the medium. The interaction profile of lys-NDs with fetal bovine serum showed formation of a protein corona, which was evaluated by size and charge distribution measurements. Uptake of lys-NDs in cervical cancer cells was analyzed by scanning transmission X-ray microscopy, flow cytometry, and confocal microscopy. Cellular uptake of diamoplexes (complex of lys-NDs with small interfering RNA) was also analyzed using flow cytometry. Results Aqueous dispersion of lys-NDs showed minimum sedimentation and remained stable over a period of 25 days. Size distributions showed good stability, remaining under 100 nm throughout the testing period. A positive zeta potential of >+20 mV indicated a preservation of surface charges. Size distribution and zeta potential changed for lys-NDs after incubation with blood serum, suggesting an interaction with biomolecules, mainly proteins, and a possible formation of a protein corona. Cellular internalization

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

PubMed

Coste, Alicia; Jager, Muriel; Chambon, Jean-Philippe; Manuel, Michaël

2016-01-01

The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

PubMed

Yang, Hong; Deng, Liwei; Li, Tingting; Shen, Xue; Yan, Jie; Zuo, Liangming; Wu, Chunhui; Liu, Yiyao

2015-12-01

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated

Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate.

PubMed

Qi, Baochang; Yu, Tiecheng; Wang, Chengxue; Wang, Tiejun; Yao, Jihang; Zhang, Xiaomeng; Deng, Pengfei; Xia, Yongning; Junger, Wolfgang G; Sun, Dahui

2016-10-03

Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose chemotherapy has been shown to improve outcome but this approach can cause many side effects. Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients. In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS cells. Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods. Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit. All data were analyzed using SPSS17.0 statistical software. Comparisons were made with t test between two groups. Treatment of human osteosarcoma U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS cells. Importantly, shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis. We found that shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis. Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.

Carbon: Nitrogen Interaction Regulates Expression of Genes Involved in N-Uptake and Assimilation in Brassica juncea L.

PubMed Central

Goel, Parul; Bhuria, Monika; Kaushal, Mamta

2016-01-01

In plants, several cellular and metabolic pathways interact with each other to regulate processes that are vital for their growth and development. Carbon (C) and Nitrogen (N) are two main nutrients for plants and coordination of C and N pathways is an important factor for maintaining plant growth and development. In the present work, influence of nitrogen and sucrose (C source) on growth parameters and expression of genes involved in nitrogen transport and assimilatory pathways was studied in B. juncea seedlings. For this, B. juncea seedlings were treated with four combinations of C and N source viz., N source alone (-Suc+N), C source alone (+Suc-N), with N and C source (+Suc+N) or without N and C source (-Suc-N). Cotyledon size and shoot length were found to be increased in seedlings, when nitrogen alone was present in the medium. Distinct expression pattern of genes in both, root and shoot tissues was observed in response to exogenously supplied N and C. The presence or depletion of nitrogen alone in the medium leads to severe up- or down-regulation of key genes involved in N-uptake and transport (BjNRT1.1, BjNRT1.8) in root tissue and genes involved in nitrate reduction (BjNR1 and BjNR2) in shoot tissue. Moreover, expression of several genes, like BjAMT1.2, BjAMT2 and BjPK in root and two genes BjAMT2 and BjGS1.1 in shoot were found to be regulated only when C source was present in the medium. Majority of genes were found to respond in root and shoot tissues, when both C and N source were present in the medium, thus reflecting their importance as a signal in regulating expression of genes involved in N-uptake and assimilation. The present work provides insight into the regulation of genes of N-uptake and assimilatory pathway in B. juncea by interaction of both carbon and nitrogen. PMID:27637072

Cellular Localization and Trafficking of the Human ABCG1 Transporter

PubMed Central

Neufeld, Edward B.; O’Brien, Katherine; Walts, Avram D.; Stonik, John A.; Demosky, Steven J.; Malide, Daniela; Combs, Christian A.; Remaley, Alan T.

2014-01-01

We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface. PMID:25405320

Xenophagic pathways and their bacterial subversion in cellular self-defense - παντα ρει - everything is in flux.

PubMed

Radomski, Nadine; Rebbig, Annica; Leonhardt, Ralf M; Knittler, Michael R

2017-11-02

Autophagy is an evolutionarily ancient and highly conserved eukaryotic mechanism that targets cytoplasmic material for degradation. Autophagic flux involves the formation of autophagosomes and their degradation by lysosomes. The process plays a crucial role in maintaining cellular homeostasis and responds to various environmental conditions. While autophagy had previously been thought to be a non-selective process, it is now clear that it can also selectively target cellular organelles, such as mitochondria (referred to as mitophagy) and/or invading pathogens (referred to as xenophagy). Selective autophagy is characterized by specific substrate recognition and requires distinct cellular adaptor proteins. Here we review xenophagic mechanisms involved in the recognition and autolysosomal or autophagolysosomal degradation of different intracellular bacteria. In this context, we also discuss a recently discovered cellular self-defense pathway, termed mito-xenophagy, which occurs during bacterial infection of dendritic cells and depends on a TNF-α-mediated metabolic switch from oxidative phosphorylation to glycolysis. Copyright © 2017 Elsevier GmbH. All rights reserved.

Impact of ocean phytoplankton diversity on phosphate uptake

PubMed Central

Lomas, Michael W.; Bonachela, Juan A.; Levin, Simon A.; Martiny, Adam C.

2014-01-01

We have a limited understanding of the consequences of variations in microbial biodiversity on ocean ecosystem functioning and global biogeochemical cycles. A core process is macronutrient uptake by microorganisms, as the uptake of nutrients controls ocean CO2 fixation rates in many regions. Here, we ask whether variations in ocean phytoplankton biodiversity lead to novel functional relationships between environmental variability and phosphate (Pi) uptake. We analyzed Pi uptake capabilities and cellular allocations among phytoplankton groups and the whole community throughout the extremely Pi-depleted western North Atlantic Ocean. Pi uptake capabilities of individual populations were well described by a classic uptake function but displayed adaptive differences in uptake capabilities that depend on cell size and nutrient availability. Using an eco-evolutionary model as well as observations of in situ uptake across the region, we confirmed that differences among populations lead to previously uncharacterized relationships between ambient Pi concentrations and uptake. Supported by novel theory, this work provides a robust empirical basis for describing and understanding assimilation of limiting nutrients in the oceans. Thus, it demonstrates that microbial biodiversity, beyond cell size, is important for understanding the global cycling of nutrients. PMID:25422472

Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles

PubMed Central

Peetla, Chiranjeevi; Rao, Kavitha S.; Labhasetwar, Vinod

2009-01-01

The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly (L-lactide) nanoparticles (RNPs), either conjugated to a TAT peptide (TAT-RNPs) or scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (π–A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir–Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP–membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift towards higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM whereas RNPs were seen anchored loosely to the

Modification in digestive processing strategies to reduce toxic trace metal uptake in a marine bivalve

DOE Office of Scientific and Technical Information (OSTI.GOV)

Decho, A.W.; Luoma, S.N.

1994-12-31

Bivalves possess two major digestion pathways for processing food particles: a rapid ``intestinal`` pathway where digestion is largely extracellular; and a slower ``glandular`` pathway where digestion is largely intracellular. The slower glandular pathway often results in more efficient absorption of carbon but also more efficient uptake of certain metals (e.g. Cr associated with bacteria). In the bivalve Potamocorbula amurensis, large portions (> 90%) of bacteria are selectively routed to the glandular pathway. This results in efficient C uptake but also efficient uptake of associated Cr. The authors further determined if prolonged exposure to Cr-contaminated bacteria would result in high Crmore » uptake by animals or whether mechanisms exist to reduce Cr exposure and uptake. Bivalves were exposed to natural food + added bacteria (with or without added Cr) for a 6-day period, then pulse-chase experiments were conducted to quantify digestive processing and % absorption efficiencies (%AE) of bacterial Cr. Bivalves compensate at low (2--5 ug/g sed) Cr by reducing overall food ingestion, while digestive processing of food remains statistically similar to controls. At high Cr (200--500 ug/g sed) there are marked decreases in % bacteria processed by glandular digestion. This results in lower overall %AE of Cr. The results suggest that bivalves under natural conditions might balance efficient carbon sequestration against avoiding uptake of potentially toxic metals associated the food.« less

Docetaxel-Loaded Self-Assembly Stearic Acid-Modified Bletilla striata Polysaccharide Micelles and Their Anticancer Effect: Preparation, Characterization, Cellular Uptake and In Vitro Evaluation.

PubMed

Guan, Qingxiang; Sun, Dandan; Zhang, Guangyuan; Sun, Cheng; Wang, Miao; Ji, Danyang; Yang, Wei

2016-12-02

Poorly soluble drugs have low bioavailability after oral administration, thereby hindering effective drug delivery. A novel drug-delivery system of docetaxel (DTX)-based stearic acid (SA)-modified Bletilla striata polysaccharides (BSPs) copolymers was successfully developed. Particle size, zeta potential, encapsulation efficiency (EE), and loading capacity (LC) were determined. The DTX release percentage in vitro was determined using high performance liquid chromatography (HPLC). The hemolysis and in vitro anticancer activity were studied. Cellular uptake and apoptotic rate were measured using flow cytometry assay. Particle size, zeta potential, EE and LC were 125.30 ± 1.89 nm, -26.92 ± 0.18 mV, 86.6% ± 0.17%, and 14.8% ± 0.13%, respectively. The anticancer activities of DTX-SA-BSPs copolymer micelles against HepG2, HeLa, SW480, and MCF-7 (83.7% ± 1.0%, 54.5% ± 4.2%, 48.5% ± 4.2%, and 59.8% ± 1.4%, respectively) were superior to that of docetaxel injection (39.2% ± 1.1%, 44.5% ± 5.3%, 38.5% ± 5.4%, and 49.8% ± 2.9%, respectively) at 0.5 μg/mL drug concentration. The DTX release percentage of DTX-SA-BSPs copolymer micelles and docetaxel injection were 66.93% ± 1.79% and 97.06% ± 1.56% in two days, respectively. Cellular uptake of DTX-FITC-SA-BSPs copolymer micelles in cells had a time-dependent relation. Apoptotic rate of DTX-SA-BSPs copolymer micelles and docetaxel injection were 73.48% and 69.64%, respectively. The SA-BSPs copolymer showed good hemocompatibility. Therefore, SA-BSPs copolymer can be used as a carrier for delivering hydrophobic drugs.

Characterization of iron uptake from transferrin by murine endothelial cells.

PubMed

Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

2000-01-01

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA.

PubMed

Al-Turkmani, M Rabie; Andersson, Charlotte; Alturkmani, Ragheed; Katrangi, Waddah; Cluette-Brown, Joanne E; Freedman, Steven D; Laposata, Michael

2008-09-01

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation.

Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways.

PubMed

Atkinson, Joshua T; Campbell, Ian; Bennett, George N; Silberg, Jonathan J

2016-12-27

The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

Cellular uptake and ex vivo urothelial penetration by oligodeoxynucleotides for optimizing treatment of transitional cell carcinoma.

PubMed

Bolenz, Christian; Trojan, Lutz; Gabriel, Ute; Honeck, Patrick; Wendt-Nordahl, Gunnar; Schaaf, Axel; Alken, Peter; Michel, Maurice Stephan

2008-10-01

To evaluate cellular uptake and urothelial penetration of oligodeoxynucleotides (ODNs) in transitional cell carcinoma (TCC) cell lines and in a porcine ex vivo model, respectively. A panel of human TCC cell lines (RT 112, HT 1197 and UM-UC3) were exposed tofluorescein-labeled ODNs. Transfection rates were assessed byfluorescence microscopy and fluorescence-activated cell sorting (FACS). Intravesical treatment with ODNs was performed in a porcine ex vivo model. Urothelial penetration was evaluated using fluorescence microscopy of cryosections. Treatment with ODNs provided transfection rates of at least 96.8% of TCC cells, irrespective of use of a transfection agent. Effective urothelial penetration by ODNs was detected when compared with controls (p = 0.0325). The addition of a liposomal transfection agent significantly increased the penetration depth, allowing affection of deep urothelial cell layers (p = 0.0082). High transfection rates of ODNs can be achieved in TCC cells. Urothelial penetration of ODNs was observed down to the deepest cell layers when a transfection agent is added, suggesting a high potential for complementing the chemoresection effects on residual tumor areas during intravesical therapy of non-muscle-invasive TCC.

Influence of Acute and Chronic Exercise on Glucose Uptake

PubMed Central

Röhling, Martin; Herder, Christian; Stemper, Theodor; Müssig, Karsten

2016-01-01

Insulin resistance plays a key role in the development of type 2 diabetes. It arises from a combination of genetic predisposition and environmental and lifestyle factors including lack of physical exercise and poor nutrition habits. The increased risk of type 2 diabetes is molecularly based on defects in insulin signaling, insulin secretion, and inflammation. The present review aims to give an overview on the molecular mechanisms underlying the uptake of glucose and related signaling pathways after acute and chronic exercise. Physical exercise, as crucial part in the prevention and treatment of diabetes, has marked acute and chronic effects on glucose disposal and related inflammatory signaling pathways. Exercise can stimulate molecular signaling pathways leading to glucose transport into the cell. Furthermore, physical exercise has the potential to modulate inflammatory processes by affecting specific inflammatory signaling pathways which can interfere with signaling pathways of the glucose uptake. The intensity of physical training appears to be the primary determinant of the degree of metabolic improvement modulating the molecular signaling pathways in a dose-response pattern, whereas training modality seems to have a secondary role. PMID:27069930

Viral Activation of Cellular Metabolism

PubMed Central

Sanchez, Erica L.; Lagunoff, Michael

2015-01-01

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol[S

PubMed Central

Iaea, David B.; Gale, Sarah E.; Bielska, Agata A.; Krishnan, Kathiresan; Fujiwara, Hideji; Jiang, Hui; Maxfield, Frederick R.; Schlesinger, Paul H.; Covey, Douglas F.; Schaffer, Jean E.; Ory, Daniel S.

2015-01-01

Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways. PMID:26497473

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with onemore » GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.« less

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

PubMed

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel method for measuring cellular antibody uptake using imaging flow cytometry reveals distinct uptake rates for two different monoclonal antibodies targeting L1.

PubMed

Hazin, John; Moldenhauer, Gerhard; Altevogt, Peter; Brady, Nathan R

2015-08-01

Monoclonal antibodies (mAbs) have emerged as a promising tool for cancer therapy. Differing approaches utilize mAbs to either deliver a drug to the tumor cells or to modulate the host's immune system to mediate tumor kill. The rate by which a therapeutic antibody is being internalized by tumor cells is a decisive feature for choosing the appropriate treatment strategy. We herein present a novel method to effectively quantitate antibody uptake of tumor cells by using image-based flow cytometry, which combines image analysis with high throughput of sample numbers and sample size. The use of this method is established by determining uptake rate of an anti-EpCAM antibody (HEA125), from single cell measurements of plasma membrane versus internalized antibody, in conjunction with inhibitors of endocytosis. The method is then applied to two mAbs (L1-9.3, L1-OV52.24) targeting the neural cell adhesion molecule L1 (L1CAM) at two different epitopes. Based on median cell population responses, we find that mAb L1-OV52.24 is rapidly internalized by the ovarian carcinoma cell line SKOV3ip while L1 mAb 9.3 is mainly retained at the cell surface. These findings suggest the L1 mAb OV52.24 as a candidate to be further developed for drug-delivery to cancer cells, while L1-9.3 may be optimized to tag the tumor cells and stimulate immunogenic cancer cell killing. Furthermore, when analyzing cell-to-cell variability, we observed L1 mAb OV52.24 rapidly transition into a subpopulation with high-internalization capacity. In summary, this novel high-content method for measuring antibody internalization rate provides a high level of accuracy and sensitivity for cell population measurements and reveals further biologically relevant information when taking into account cellular heterogeneity. Copyright © 2015 Elsevier B.V. All rights reserved.

Celllular Uptake and Clearance of TIO2 Nanoparticles

EPA Science Inventory

Differential rates of cellular uptake and clearance of engineered nanomaterials may influence the propensity for tissue accumulation under chronic exposure conditions. A retinal pigment epithelial cell line (ARPE-19) was used to investigate 1) if Ti02 (Degussa, P25) nanoparticles...

Polymethoxyflavonoids tangeretin and nobiletin increase glucose uptake in murine adipocytes.

PubMed

Onda, Kenji; Horike, Natsumi; Suzuki, Tai-ichi; Hirano, Toshihiko

2013-02-01

Tangeretin and nobiletin are polymethoxyflavonoids that are contained in citrus fruits. Polymethoxyflavonoids are reported to have several biological functions including anti-inflammatory, anti-atherogenic, or anti-diabetic effects. However, whether polymethoxyflavonoids directly affect glucose uptake in tissues is not well understood. In the current study, we investigated whether tangeretin and nobiletin affect glucose uptake in insulin target cells such as adipocytes. We observed that treatment with tangeretin or nobiletin significantly increased the uptake of [(3) H]-deoxyglucose in differentiated 3T3-F442A adipocytes in a concentration-dependent manner. Data showed that phosphatidyl inositol 3 kinase, Akt1/2, and the protein kinase A pathways were involved in the increase in glucose uptake induced by polymethoxyflavonoids. These data suggest that the anti-diabetic action of polymethoxyflavonoids is partly exerted via these signaling pathways in insulin target tissues. Copyright © 2012 John Wiley & Sons, Ltd.

The mechanism of zinc uptake by cultured rat liver cells.

PubMed Central

Taylor, J A; Simons, T J

1994-01-01

1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898

A missense mutation in TCN2 is associated with decreased risk for congenital heart defects and may increase cellular uptake of vitamin B12 via Megalin.

PubMed

Li, Peiqiang; Huang, Lijuan; Zheng, Yufang; Pan, Xuedong; Peng, Rui; Jiang, Yueming; Finnell, Richard H; Li, Haijie; Qiao, Bin; Wang, Hong-Yan

2017-08-15

Deregulation of folate and vitamin B12 (VB12) metabolism contributes to the risk of congenital heart defects (CHDs). Transcobalamin (TCN2) is essential for transporting VB12 from blood to cells as TCN2-bound VB12 (holo-TC) is the only form for somatic cellular uptake. In this study, we performed an association study between common polymorphisms in 46 one carbon metabolism genes and CHD in 412 CHDs and 213 controls. Only two significant association signals in coding regions were identified: FTCD c.1470C>T & TCN2 c.230A>T. The only missense mutation, TCN2 c.230A>T, was further validated in 412 CHDs and 1177 controls. TCN2 c.230T is significantly associated with reduced CHD risk in North Chinese (odds ratio = 0.67, P = 4.62e-05), compared with the 230A allele. Interestingly, the mean level of plasma holo-TC in women with the TA genotype was 1.77-fold higher than that in women with the AA genotype. Further analysis suggested that c.230A>T enhanced the cellular uptake of holo-TC via the LRP2 receptor. Our results determined that a functional polymorphism in TCN2 contributes to the prevalence of CHDs. TCN2 c.230A>T is significantly associated with a reduced CHD risk, likely due to TCN2 c.230T improving the interaction between holo-TC and its LRP2 receptor.

A missense mutation in TCN2 is associated with decreased risk for congenital heart defects and may increase cellular uptake of vitamin B12 via Megalin

PubMed Central

Zheng, Yufang; Pan, Xuedong; Peng, Rui; Jiang, Yueming; Finnell, Richard H.; Li, Haijie; Qiao, Bin; Wang, Hong-Yan

2017-01-01

Deregulation of folate and vitamin B12 (VB12) metabolism contributes to the risk of congenital heart defects (CHDs). Transcobalamin (TCN2) is essential for transporting VB12 from blood to cells as TCN2-bound VB12 (holo-TC) is the only form for somatic cellular uptake. In this study, we performed an association study between common polymorphisms in 46 one carbon metabolism genes and CHD in 412 CHDs and 213 controls. Only two significant association signals in coding regions were identified: FTCD c.1470C>T & TCN2 c.230A>T. The only missense mutation, TCN2 c.230A>T, was further validated in 412 CHDs and 1177 controls. TCN2 c.230T is significantly associated with reduced CHD risk in North Chinese (odds ratio = 0.67, P = 4.62e-05), compared with the 230A allele. Interestingly, the mean level of plasma holo-TC in women with the TA genotype was 1.77-fold higher than that in women with the AA genotype. Further analysis suggested that c.230A>T enhanced the cellular uptake of holo-TC via the LRP2 receptor. Our results determined that a functional polymorphism in TCN2 contributes to the prevalence of CHDs. TCN2 c.230A>T is significantly associated with a reduced CHD risk, likely due to TCN2 c.230T improving the interaction between holo-TC and its LRP2 receptor. PMID:28903415

Distinct pathways of humoral and cellular immunity induced with the mucosal administration of a nanoemulsion adjuvant.

PubMed

Bielinska, Anna U; Makidon, Paul E; Janczak, Katarzyna W; Blanco, Luz P; Swanson, Benjamin; Smith, Douglas M; Pham, Tiffany; Szabo, Zsuzsanna; Kukowska-Latallo, Jolanta F; Baker, James R

2014-03-15

Nasal administration of an oil-in-water nanoemulsion (NE) adjuvant W805EC produces potent systemic and mucosal, Th-1- and Th-17-balanced cellular responses. However, its molecular mechanism of action has not been fully characterized and is of particular interest because NE does not contain specific ligands for innate immune receptors. In these studies, we demonstrate that W805EC NE adjuvant activates innate immunity, induces specific gene transcription, and modulates NF-κB activity via TLR2 and TLR4 by a mechanism that appears to be distinct from typical TLR agonists. Nasal immunization with NE-based vaccine showed that the TLR2, TLR4, and MyD88 pathways and IL-12 and IL-12Rβ1 expression are not required for an Ab response, but they are essential for the induction of balanced Th-1 polarization and Th-17 cellular immunity. NE adjuvant induces MHC class II, CD80, and CD86 costimulatory molecule expression and dendritic cell maturation. Further, upon immunization with NE, adjuvant mice deficient in the CD86 receptor had normal Ab responses but significantly reduced Th-1 cellular responses, whereas animals deficient in both CD80 and CD86 or lacking CD40 failed to produce either humoral or cellular immunity. Overall, our data show that intranasal administration of Ag with NE induces TLR2 and TLR4 activation along with a MyD88-independent Ab response and a MyD88-dependent Th-1 and Th-17 cell-mediated immune response. These findings suggest that the unique properties of NE adjuvant may offer novel opportunities for understanding previously unrecognized mechanisms of immune activation important for generating effective mucosal and systemic immune responses.

Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

PubMed

Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

2009-01-19

A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

Induction of cellular and molecular immunomodulatory pathways by vitamin A and Flavonoids

PubMed Central

Patel, Sapna; Vajdy, Michael

2016-01-01

Introduction A detailed study of reports on the immunomodulatory properties of vitamin A and select flavonoids may pave the way for using these natural compounds or compounds with similar structures in novel drug and vaccine designs against infectious and autoimmune diseases and cancers. Areas Covered Intracellular transduction pathways, cellular differentiation and functional immunomodulatory responses have been reviewed. The reported studies encompass in vitro, in vivo preclinical and clinical studies that address the role of Vitamin A and select flavonoids in induction of innate and adaptive B and T cell responses, including TH1, TH2 and Treg. Expert Opinion While the immunomodulatory role of vitamin A, and related compounds, is well-established in many preclinical studies, its role in humans has begun to gain wider acceptance. In contrast, the role of flavonoids is mostly controversial in clinical trials, due to the diversity of the various classes of these compounds, and possibly due to the purity and the selected doses of the compounds. However, current preclinical and clinical studies warrant further detailed studies of these promising immuno-modulatory compounds. PMID:26185959

Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism

PubMed Central

Hamdi, M M; Mutungi, G

2011-01-01

Abstract Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression. PMID:21606113

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation.

PubMed

Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

2016-11-25

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation*

PubMed Central

Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

2016-01-01

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. PMID:27756839

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

PubMed

Anzinger, Joshua J; Chang, Janet; Xu, Qing; Buono, Chiara; Li, Yifu; Leyva, Francisco J; Park, Bum-Chan; Greene, Lois E; Kruth, Howard S

2010-10-01

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Cellular death, reactive oxygen species (ROS) and diabetic complications.

PubMed

Volpe, Caroline Maria Oliveira; Villar-Delfino, Pedro Henrique; Dos Anjos, Paula Martins Ferreira; Nogueira-Machado, José Augusto

2018-01-25

Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Several signaling pathways can be altered by having hyperglycemia in different tissues, producing oxidative stress, the formation of advanced glycation end products (AGEs), as well as the secretion of the pro-inflammatory cytokines and cellular death (pathological autophagy and/or apoptosis). However, the signaling pathways that are directly triggered by hyperglycemia appear to have a pivotal role in diabetic complications due to the production of reactive oxygen species (ROS), oxidative stress, and cellular death. The present review will discuss the role of cellular death in diabetic complications, and it will suggest the cause and the consequences between the hyperglycemia-induced signaling pathways and cell death. The signaling pathways discussed in this review are to be described step-by-step, together with their respective inhibitors. They involve diacylglycerol, the activation of protein kinase C (PKC) and NADPH-oxidase system, and the consequent production of ROS. This was initially entitled the "dangerous metabolic route in diabetes". The historical usages and the recent advancement of new drugs in controlling possible therapeutical targets have been highlighted, in order to evaluate the evolution of knowledge in this sensitive area. It has recently been shown that the metabolic responses to stimuli (i.e., hyperglycemia) involve an integrated network of signaling pathways, in order to define the exact responses. Certain new drugs have been experimentally tested-or suggested and proposed-for their ability to modulate the possible biochemical therapeutical targets for the downregulation of retinopathy, nephropathy, neuropathy, heart disease, angiogenesis, oxidative stress, and cellular death. The aim of this study was to critically and didactically evaluate the exact steps of these signaling pathways and hence mark the indicated sites for the actions of such

Recombinant glucose uptake system

DOEpatents

Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

1997-01-01

Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

PubMed Central

Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

2016-01-01

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

PubMed

Kraehling, Jan R; Chidlow, John H; Rajagopal, Chitra; Sugiyama, Michael G; Fowler, Joseph W; Lee, Monica Y; Zhang, Xinbo; Ramírez, Cristina M; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L; Fernández-Hernando, Carlos; Sessa, William C

2016-11-21

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Mathematical Modeling and Experimental Validation of Nanoemulsion-Based Drug Transport across Cellular Barriers.

PubMed

Kadakia, Ekta; Shah, Lipa; Amiji, Mansoor M

2017-07-01

Nanoemulsions have shown potential in delivering drug across epithelial and endothelial cell barriers, which express efflux transporters. However, their transport mechanisms are not entirely understood. Our goal was to investigate the cellular permeability of nanoemulsion-encapsulated drugs and apply mathematical modeling to elucidate transport mechanisms and sensitive nanoemulsion attributes. Transport studies were performed in Caco-2 cells, using fish oil nanoemulsions and a model substrate, rhodamine-123. Permeability data was modeled using a semi-mechanistic approach, capturing the following cellular processes: endocytotic uptake of the nanoemulsion, release of rhodamine-123 from the nanoemulsion, efflux and passive permeability of rhodamine-123 in aqueous solution. Nanoemulsions not only improved the permeability of rhodamine-123, but were also less sensitive to efflux transporters. The model captured bidirectional permeability results and identified sensitive processes, such as the release of the nanoemulsion-encapsulated drug and cellular uptake of the nanoemulsion. Mathematical description of cellular processes, improved our understanding of transport mechanisms, such as nanoemulsions don't inhibit efflux to improve drug permeability. Instead, their endocytotic uptake, results in higher intracellular drug concentrations, thereby increasing the concentration gradient and transcellular permeability across biological barriers. Modeling results indicated optimizing nanoemulsion attributes like the droplet size and intracellular drug release rate, may further improve drug permeability.

Interaction with culture medium components, cellular uptake and intracellular distribution of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts.

PubMed

Sabbioni, Enrico; Fortaner, Salvador; Farina, Massimo; Del Torchio, Riccardo; Petrarca, Claudia; Bernardini, Giovanni; Mariani-Costantini, Renato; Perconti, Silvia; Di Giampaolo, Luca; Gornati, Rosalba; Di Gioacchino, Mario

2014-02-01

The mechanistic understanding of nanotoxicity requires the physico-chemical characterisation of nanoparticles (NP), and their comparative investigation relative to the corresponding ions and microparticles (MP). Following this approach, the authors studied the dissolution, interaction with medium components, bioavailability in culture medium, uptake and intracellular distribution of radiolabelled Co forms (CoNP, CoMP and Co(2+)) in Balb/3T3 mouse fibroblasts. Co(2+) first saturates the binding sites of molecules in the extracellular milieu (e.g., albumin and histidine) and on the cell surface. Only after saturation, Co(2+) is actively uptaken. CoNP, instead, are predicted to be internalised by endocytosis. Dissolution of Co particles allows the formation of Co compounds (CoNP-rel), whose mechanism of cellular internalisation is unknown. Co uptake (ranking CoMP > CoNP > Co(2+)) reached maximum at 4 h. Once inside the cell, CoNP spread into the cytosol and organelles. Consequently, massive amounts of Co ions and CoNP-rel can reach subcellular compartments normally unexposed to Co(2+). This could explain the fact that the nuclear and mitochondrial Co concentrations resulted significantly higher than those obtained with Co(2+).

Fluorophore:dendrimer ratio impacts cellular uptake and intracellular fluorescence lifetime.

PubMed

Dougherty, Casey A; Vaidyanathan, Sriram; Orr, Bradford G; Banaszak Holl, Mark M

2015-02-18

G5-NH2-TAMRAn (n = 1-4, 5+, and 1.5(avg)) were prepared with n = 1-4 as a precise dye:dendrimer ratio, 5+ as a mixture of dendrimers with 5 or more dye per dendrimer, and 1.5(avg) as a Poisson distribution of dye:dendrimer ratios with a mean of 1.5 dye per dendrimer. The absorption intensity increased sublinearly with n whereas the fluorescence emission and lifetime decreased with an increasing number of dyes per dendrimer. Flow cytometry was employed to quantify uptake into HEK293A cells. Dendrimers with 2-4 dyes were found to have greater uptake than dendrimer with a single dye. Fluorescence lifetime imaging microscopy (FLIM) showed that the different dye:dendrimer ratio alone was sufficient to change the fluorescence lifetime of the material observed inside cells. We also observed that the lifetime of G5-NH2-TAMRA5+ increased when present in the cell as compared to solution. However, cells treated with G5-NH2-TAMRA1.5(avg) did not exhibit the high lifetime components present in G5-NH2-TAMRA1 and G5-NH2-TAMRA5+. In general, the effects of the dye:dendrimer ratio on fluorescence lifetime were of similar magnitude to environmentally induced lifetime shifts.

The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

PubMed

Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

1995-10-24

Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

Involvement of multiple cellular pathways in regulating resistance to tamoxifen in BIK-suppressed MCF-7 cells.

PubMed

Viedma-Rodríguez, Rubí; Ruiz Esparza-Garrido, Ruth; Baiza-Gutman, Luis Arturo; Velázquez-Flores, Miguel Ángel; García-Carrancá, Alejandro; Salamanca-Gómez, Fabio; Arenas-Aranda, Diego

2015-09-01

Majority of women with estrogen receptor (ER)-positive breast cancers initially respond to hormone therapies such as tamoxifen (TAM; antagonist of estrogen). However, many tumors eventually become resistant to TAM. Therefore, understanding the various cellular components involved in causing resistance to TAM is of paramount importance in designing novel entities for efficacious hormone therapy. Previously, we found that suppression of BIK gene expression induced TAM resistance in MCF-7 breast cancer cells. In order to understand the response of these cells to TAM and its association with resistance, a microarray analysis of gene expression was performed in the BIK-suppressed MCF-7 cells and compared it to the TAM-only-treated cells (controls). Several genes participating in various cellular pathways were identified. Molecules identified in the drug resistance pathway were 14-3-3z or YWHAZ, WEE1, PRKACA, NADK, and HSP90AA 1. Further, genes involved in cell cycle control, apoptosis, and cell proliferation were also found differentially expressed in these cells. Transcriptional and translational analysis of key molecules such as STAT2, AKT 3, and 14-3-3z revealed similar changes at the messenger RNA (mRNA) as well as at the protein level. Importantly, there was no cytotoxic effect of TAM on BIK-suppressed MCF-7 cells. Further, these cells were not arrested at the G0-G1 phase of the cell cycle although 30 % of BIK-suppressed cells were arrested at the G2 phase of the cycle on TAM treatment. Furthermore, we found a relevant interaction between 14-3-3z and WEE1, suggesting that the cytotoxic effect of TAM was prevented in BIK-suppressed cells because this interaction leads to transitory arrest in the G2 phase leading to the repair of damaged DNA and allowing the cells to proliferate.

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials.

PubMed

Quach, Quang Huy; Kah, James Chen Yong

2017-04-01

The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.

Central obesity, type 2 diabetes and insulin: exploring a pathway full of thorns

PubMed Central

Papakyriakou, Panagiotis; Panagiotou, Themistoklis N.

2015-01-01

The prevalence of type 2 diabetes (T2D) is rapidly increasing. This is strongly related to the contemporary lifestyle changes that have resulted in increased rates of overweight individuals and obesity. Central (intra-abdominal) obesity is observed in the majority of patients with T2D. It is associated with insulin resistance, mainly at the level of skeletal muscle, adipose tissue and liver. The discovery of macrophage infiltration in the abdominal adipose tissue and the unbalanced production of adipocyte cytokines (adipokines) was an essential step towards novel research perspectives for a better understanding of the molecular mechanisms governing the development of insulin resistance. Furthermore, in an obese state, the increased cellular uptake of non-esterified fatty acids is exacerbated without any subsequent β-oxidation. This in turn contributes to the accumulation of intermediate lipid metabolites that cause defects in the insulin signaling pathway. This paper examines the possible cellular mechanisms that connect central obesity with defects in the insulin pathway. It discusses the discrepancies observed from studies organized in cell cultures, animal models and humans. Finally, it emphasizes the need for therapeutic strategies in order to achieve weight reduction in overweight and obese patients with T2D. PMID:26170839

Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

PubMed Central

Varanasi, Lakshman; Hosler, Jonathan P.

2011-01-01

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (−)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O2 consumption can be used to compare the rate and functional pKa of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (−) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake. PMID:22023935

Cellular uptake of glucoheptoamidated poly(amidoamine) PAMAM G3 dendrimer with amide-conjugated biotin, a potential carrier of anticancer drugs.

PubMed

Uram, Łukasz; Szuster, Magdalena; Filipowicz, Aleksandra; Zaręba, Magdalena; Wałajtys-Rode, Elżbieta; Wołowiec, Stanisław

2017-01-15

In search for soluble derivatives of PAMAM dendrimers as potential carriers for hydrophobic drugs, the conjugates of PAMAM G3 with biotin, further converted into glycodendrimer with d-glucoheptono-1,4-lactone, were prepared. Polyamidoamine dendrimer (PAMAM) of third generation, G3 was functionalized with four biotin equivalents covalently attached to terminal amine nitrogens via amide bond G3 4B . The remaining 28 amine groups were blocked by glucoheptoamide substituents (gh) to give G3 4B28gh or with one fluorescein equivalent (attached by reaction of G3 4B with fluorescein isothiocyanate, FITC) via thiourea bond as FITC followed by exhaustive glucoheptoamidation to get G3 4B27gh1F . As a control the G3 substituted totally with 32 glucoheptoamide residues, G3 gh and its fluorescein labeled analogue G3 31gh1F were synthesized. The glucoheptoamidation of PAMAM G0 dendrimer with glucoheptono-1,4-lactone was performed in order to fully characterize the 1 H NMR spectra of glucoheptoamidated PAMAM dendrimers and to control the derivatization of G3 with glucoheptono-1,4-lactone. Another two derivatives of G3, namely G3 4B28gh1F' and G3 32ghF' , with ester bonded fluorescein were also obtained. Biological properties of obtained dendrimer conjugates were estimated in vitro with human cell lines: normal fibroblast (BJ) and two cancer glioblastoma (U-118 MG) and squamous carcinoma (SCC-15), including cytotoxicity by reduction of XTT and neutral red (NR) assays. Cellular uptake of dendrimer conjugates was evaluated with confocal microscopy. Obtained results confirmed, that biotinylated bioconjugates have always lower cytotoxicity and 3-4 times higher cellular uptake than non-biotinylated dendrimer conjugates in all cell lines. Comparison of various cell lines revealed different dose-dependent cell responses and the lower cytotoxicity of examined dendrimer conjugates for normal fibroblasts and squamous carcinoma, as compared with much higher cytotoxic effects seen in

Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake.

PubMed

Ahire, Jayshree H; Behray, Mehrnaz; Webster, Carl A; Wang, Qi; Sherwood, Victoria; Saengkrit, Nattika; Ruktanonchai, Uracha; Woramongkolchai, Noppawan; Chao, Yimin

2015-08-26

The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Shengke; Xie, Ruohan; Wang, Haixin

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cdmore » sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.« less

Polyamine Uptake in Carrot Cell Cultures 1

PubMed Central

Pistocchi, Rossella; Bagni, Nello; Creus, José A.

1987-01-01

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca2+. The effects of Ca2+ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca2+ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca2+ concentration range between 10 micromolar and 1 millimolar. La3+ nullified the stimulatory effect of 10 micromolar Ca2+ on putrescine uptake and that of 1 millimolar Ca2+ on spermidine uptake. La3+ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule. PMID:16665446

Identification of an iron permease, cFTR1, in cyanobacteria involved in the iron reduction/re-oxidation uptake pathway.

PubMed

Xu, Ning; Qiu, Guo-Wei; Lou, Wen-Jing; Li, Zheng-Ke; Jiang, Hai-Bo; Price, Neil M; Qiu, Bao-Sheng

2016-12-01

Cyanobacteria are globally important primary producers and abundant in many iron-limited aquatic environments. The ways in which they take up iron are largely unknown, but reduction of Fe 3+ is an important step in the process. Here we report a special iron permease in Synechocystis, cFTR1, that is required for Fe 3+ uptake following Fe 2+ re-oxidation. The expression of cFTR1 is induced by iron starvation, and a mutant lacking the gene is abnormally sensitive to iron starvation. The cFTR1 protein localizes to the plasma membrane and contains the iron-binding motif "REXXE". Point-directed mutagenesis of the REXXE motif results in a sensitivity to Fe-deficiency. Measurements of iron ( 55 Fe) uptake rate show that cFTR1 takes up Fe 3+ rather than Fe 2+ . The function of cFTR1 in Synechocystis could be genetically complemented by the iron permease, Ftr1p, of Saccharomyces cerevisiae, that is known to transport Fe 3+ produced by the oxidation of Fe 2+ via a multicopper oxidase. Unlike yeast Ftr1p, cyanobacterial cFTR1 probably obtains Fe 3+ primarily from the oxidation of Fe 2+ by oxygen. Growth assays show that the cFTR1 is required during oxygenic, photoautotrophic growth but not when oxygen production is inhibited during photoheterotrophic growth. In cyanobacteria, iron reduction/re-oxidation uptake pathway may represent their adaptation to oxygenated environments. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Hypoxia modifies nuclear calcium uptake pathways in the cerebral cortex of the guinea-pig fetus.

PubMed

Zanelli, S A; Spandou, E; Mishra, O P; Delivoria-Papadopoulos, M

2005-01-01

Nuclear Ca2+ signals are thought to play a critical role in the initiation and progression of programmed cell death. The present study tests the hypothesis that hypoxia alters nuclear Ca2+ transport pathways and leads to an increase in nuclear Ca(2+)-influx in cerebral cortical neuronal nuclei. To test this hypothesis the effect of tissue hypoxia on high affinity Ca(2+)-ATPase activity and the binding characteristics of inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) receptors were studied in neuronal nuclei from the cerebral cortex of guinea-pig fetuses. Results show increased high-affinity Ca(2+)-ATPase activity (nmol/mg protein/h) in the hypoxic group 969.7+/-79 as compared with 602.4+/-90.9 in the normoxic group, P<0.05. The number of IP3 receptors (Bmax, fmol/mg protein) increased from 61+/-21 in the normoxic group to 164+/-49 in the hypoxic group, P<0.05. K(d) values did not change following hypoxia. In contrast, IP4 receptor Bmax (fmol/mg protein) and K(d) (nM) values increased from 360+/-32 in the normoxic group to 626+/-136 in the hypoxic group (P<0.001) and, from 26+/-1 in the normoxic group to 61+/-9 in the hypoxic group (P<0.001), respectively. 45Ca(2+)-influx (pmol/mg protein) significantly increased from 6.3+/-1.9 in the normoxic group to 10.9+/-1.1 the hypoxic group (P<0.001). The data show that hypoxia modifies nuclear Ca2+ transport pathways and results in increased nuclear Ca(2+)-influx. We speculate that hypoxia increases nuclear Ca2+ uptake from the cytoplasm to the nucleoplasm, resulting in increased transcription of proapoptotic genes and subsequent activation of programmed cell death pathways.

Oligodeoxynucleotide nanostructure formation in the presence of polypropyleneimine dendrimers and their uptake in breast cancer cells

NASA Astrophysics Data System (ADS)

Chen, Alex M.; Santhakumaran, Latha M.; Nair, Sandhya K.; Amenta, Peter S.; Thomas, Thresia; He, Huixin; Thomas, T. J.

2006-11-01

We studied the efficacy of five generations of polypropyleneimine (PPI) dendrimer to provoke nanostructure formation from a 21-nucleotide antisense oligodeoxynucleotide (ODN). Nanostructure formation was observed with all generations of dendrimer by light scattering and microscopic techniques. The efficacy of the dendrimers increased with generation number. Atomic force microscopy (AFM) was used to study the morphology of the structures at different condensation stages. Based on the observed nanostructures, we propose a zipping condensation mechanism, which is very different from the condensation pathways of high molecular weight DNA polymers. Electron microscopy showed the presence of toroidal nanoparticles. Confocal microscopic analysis showed that the nanostructures formed with G-4 and G-5 dendrimers could undergo facile cellular uptake in a breast cancer cell line, MDA-MB-231, whereas nanostructures formed with G-1 to G-3 dendrimers lacked this ability. Nanoparticles formed with G-1 to G-3 dendrimers showed significantly lower zeta potential (5.2-6.5 mV) than those (12-18 mV) of particles formed with G-4 and G-5 dendrimers. These results show that the structure and charge density of the dendrimers are important in ODN nanoparticle formation and cellular transport and that G-4 and G-5 dendrimers are useful in cellular delivery of antisense ODN.

Cell Proliferation, Reactive Oxygen and Cellular Glutathione

PubMed Central

Day, Regina M.; Suzuki, Yuichiro J.

2005-01-01

A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth. PMID:18648617

Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes.

PubMed

Makino-Okamura, Chieko; Niki, Yoko; Takeuchi, Seiji; Nishigori, Chikako; Declercq, Lieve; Yaroch, Daniel B; Saito, Naoaki

2014-11-01

To gain insight for the role of mast cell-produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin-treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2 ) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti-inflammation role in epidermis of human skin. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Vacuolar amino acid transporter Avt5p is responsible for lithium uptake in Schizosaccharomyces pombe.

PubMed

Iwaki, Tomoko; Sekito, Takayuki; Kakinuma, Yoshimi

2010-01-01

The fission yeast Schizosaccharomyces pombe was sensitive to salinity; cell growth was stopped by 0.5 M NaCl and by 10 mM LiCl. The avt5+ gene encodes a vacuolar transporter with a broad specificity for amino acids. We found that the avt5Delta mutant became highly tolerant of Li+ and Na+ in growth. Concanamycin A-sensitive Li+ uptake as well as cellular Li+ content was lower in the avt5 mutant, suggesting a role of Avt5p in cellular uptake of toxic Li+.

Variable phosphorus uptake rates and allocation across microbial groups in the oligotrophic Gulf of Mexico.

PubMed

Popendorf, Kimberly J; Duhamel, Solange

2015-10-01

Microbial uptake of dissolved phosphorus (P) is an important lever in controlling both microbial production and the fate and cycling of marine P. We investigated the relative role of heterotrophic bacteria and phytoplankton in P cycling by measuring the P uptake rates of individual microbial groups (heterotrophic bacteria and the phytoplankton groups Synechococcus, Prochlorococcus and picoeukaryotic phytoplankton) in the P-depleted Gulf of Mexico. Phosphorus uptake rates were measured using incubations with radiolabelled phosphate and adenosine triphosphate coupled with cell sorting flow cytometry. We found that heterotrophic bacteria were the dominant consumers of P on both a biomass basis and a population basis. Biovolume normalized heterotrophic bacteria P uptake rate per cell (amol P μm(-3) h(-1)) was roughly an order of magnitude greater than phytoplankton uptake rates, and heterotrophic bacteria were responsible for generally greater than 50% of total picoplankton P uptake. We hypothesized that this variation in uptake rates reflects variation in cellular P allocation strategies, and found that, indeed, the fraction of cellular P uptake utilized for phospholipid production was significantly higher in heterotrophic bacteria compared with cyanobacterial phytoplankton. These findings indicate that heterotrophic bacteria have a uniquely P-oriented physiology and play a dominant role in cycling dissolved P. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Cellular Response to Ionizing Radiation: A MicroRNA Story

PubMed Central

Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

2012-01-01

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

Genetic Dominance & Cellular Processes

ERIC Educational Resources Information Center

Seager, Robert D.

2014-01-01

In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

Cellular mechanism of oral absorption of solidified polymer micelles.

PubMed

Abramov, Eva; Cassiola, Flavia; Schwob, Ouri; Karsh-Bluman, Adi; Shapero, Mara; Ellis, James; Luyindula, Dema; Adini, Irit; D'Amato, Robert J; Benny, Ofra

2015-11-01

Oral delivery of poorly soluble and permeable drugs represents a significant challenge in drug development. The oral delivery of drugs remains to be the ultimate route of any drugs. However, in many cases, drugs are not absorbed well in the gastrointestinal tract, or they lose their activity. Polymer micelles were recognized as an effective carrier system for drug encapsulation, and are now studied as a vehicle for oral delivery of insoluble compounds. We characterized the properties of monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles, and visualized their internalization in mouse small intestine. Using Caco-2 cells as a cellular model, we studied the kinetics of particle uptake, their transport, and the molecular mechanism of their intestinal absorption. Moreover, by inhibiting specific endocytosis pathways, pharmacologically and genetically, we found that mPEG-PLA nanoparticle endocytosis is mediated by clathrin in an energy-dependent manner, and that the low-density lipoprotein receptor is involved. Many current drugs used are non-water soluble and indeed, the ability to deliver these drugs via the gastrointestinal tract remains the holy grail for many researchers. The authors in this paper developed monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles as a drug nanocarrier, and studied the mechanism of uptake across intestinal cells. The findings should improve our current understanding and point to the development of more nanocarriers. Copyright © 2015 Elsevier Inc. All rights reserved.

Emerging role of Hippo signalling pathway in bladder cancer.

PubMed

Xia, Jianling; Zeng, Ming; Zhu, Hua; Chen, Xiangjian; Weng, Zhiliang; Li, Shi

2018-01-01

Bladder cancer (BC) is one of the most common cancers worldwide with a high progression rate and poor prognosis. The Hippo signalling pathway is a conserved pathway that plays a crucial role in cellular proliferation, differentiation and apoptosis. Furthermore, dysregulation and/or malfunction of the Hippo pathway is common in various human tumours, including BC. In this review, an overview of the Hippo pathway in BC and other cancers is presented. We focus on recent data regarding the Hippo pathway, its network and the regulation of the downstream co-effectors YAP1/TAZ. The core components of the Hippo pathway, which induce BC stemness acquisition, metastasis and chemoresistance, will be emphasized. Additional research on the Hippo pathway will advance our understanding of the mechanism of BC as well as the development and progression of other cancers and may be exploited therapeutically. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

PubMed Central

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake.

PubMed

Mirshafiee, Vahid; Kim, Raehyun; Park, Soyun; Mahmoudi, Morteza; Kraft, Mary L

2016-01-01

Nanoparticles (NPs) are functionalized with targeting ligands to enable selectively delivering drugs to desired locations in the body. When these functionalized NPs enter the blood stream, plasma proteins bind to their surfaces, forming a protein corona that affects NP uptake and targeting efficiency. To address this problem, new strategies for directing the formation of a protein corona that has targeting capabilities are emerging. Here, we have investigated the feasibility of directing corona composition to promote targeted NP uptake by specific types of cells. We used the well-characterized process of opsonin-induced phagocytosis by macrophages as a simplified model of corona-mediated NP uptake by a desired cell type. We demonstrate that pre-coating silica NPs with gamma-globulins (γ-globulins) produced a protein corona that was enriched with opsonins, such as immunoglobulins. Although immunoglobulins are ligands that bind to receptors on macrophages and elicit phagocytois, the opsonin-rich protein corona did not increase NP uptake by macrophage RAW 264.7 cells. Immunolabeling experiments indicated that the binding of opsonins to their target cell surface receptors was impeded by other proteins in the corona. Thus, corona-mediated NP targeting strategies must optimize both the recruitment of the desired plasma proteins as well as their accessibility and orientation in the corona layer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways.

PubMed

Swedan, Samer; Musiyenko, Alla; Barik, Sailen

2009-10-01

Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKKepsilon, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKKepsilon proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKKepsilon levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKKepsilon levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKKepsilon and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.

Alzheimer's amyloid-β oligomers rescue cellular prion protein induced tau reduction via the Fyn pathway.

PubMed

Chen, Rong-Jie; Chang, Wei-Wei; Lin, Yu-Chun; Cheng, Pei-Lin; Chen, Yun-Ru

2013-09-18

Amyloid-β (Aβ) and tau are the pathogenic hallmarks in Alzheimer's disease (AD). Aβ oligomers are considered the actual toxic entities, and the toxicity relies on the presence of tau. Recently, Aβ oligomers have been shown to specifically interact with cellular prion protein (PrP(C)) where the role of PrP(C) in AD is still not fully understood. To investigate the downstream mechanism of PrP(C) and Aβ oligomer interaction and their possible relationships to tau, we examined tau expression in human neuroblastoma BE(2)-C cells transfected with murine PrP(C) and studied the effect under Aβ oligomer treatment. By Western blotting, we found that PrP(C) overexpression down-regulated tau protein and Aβ oligomer binding alleviated the tau reduction induced by wild type but not M128V PrP(C), the high AD risk polymorphic allele in human prion gene. PrP(C) lacking the Aβ oligomer binding site was incapable of rescuing the level of tau reduction. Quantitative RT-PCR showed the PrP(C) effect was attributed to tau reduction at the transcription level. Treatment with Fyn pathway inhibitors, Fyn kinase inhibitor PP2 and MEK inhibitor U0126, reversed the PrP(C)-induced tau reduction and Aβ oligomer treatment modulated Fyn kinase activity. The results suggested Fyn pathway regulated Aβ-PrP(C)-tau signaling. Overall, our results demonstrated that PrP(C) down-regulated tau via the Fyn pathway and the effect can be regulated by Aβ oligomers. Our study facilitated the understanding of molecular mechanisms among PrP(C), tau, and Aβ oligomers.

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

PubMed Central

Yun, Bo; Azad, Mohammad A. K.; Nowell, Cameron J.; Nation, Roger L.; Thompson, Philip E.; Roberts, Kade D.

2015-01-01

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. PMID:26392495

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach.

PubMed

Triboulet, Sarah; Aude-Garcia, Catherine; Armand, Lucie; Gerdil, Adèle; Diemer, Hélène; Proamer, Fabienne; Collin-Faure, Véronique; Habert, Aurélie; Strub, Jean-Marc; Hanau, Daniel; Herlin, Nathalie; Carrière, Marie; Van Dorsselaer, Alain; Rabilloud, Thierry

2014-06-07

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

Cellular compartmentalization of secondary metabolism

USDA-ARS?s Scientific Manuscript database

Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors sh...

Intersection of autophagy with pathways of antigen presentation.

PubMed

Patterson, Natalie L; Mintern, Justine D

2012-12-01

Traditionally, macroautophagy (autophagy) is viewed as a pathway of cell survival. Autophagy ensures the elimination of damaged or unwanted cytosolic components and provides a source of cellular nutrients during periods of stress. Interestingly, autophagy can also directly intersect with, and impact, other major pathways of cellular function. Here, we will review the contribution of autophagy to pathways of antigen presentation. The autophagy machinery acts to modulate both MHCI and MHCII antigen presentation. As such autophagy is an important participant in pathways that elicit host cell immunity and the elimination of infectious pathogens.

Muscle redox signalling pathways in exercise. Role of antioxidants.

PubMed

Mason, Shaun A; Morrison, Dale; McConell, Glenn K; Wadley, Glenn D

2016-09-01

Recent research highlights the importance of redox signalling pathway activation by contraction-induced reactive oxygen species (ROS) and nitric oxide (NO) in normal exercise-related cellular and molecular adaptations in skeletal muscle. In this review, we discuss some potentially important redox signalling pathways in skeletal muscle that are involved in acute and chronic responses to contraction and exercise. Specifically, we discuss redox signalling implicated in skeletal muscle contraction force, mitochondrial biogenesis and antioxidant enzyme induction, glucose uptake and muscle hypertrophy. Furthermore, we review evidence investigating the impact of major exogenous antioxidants on these acute and chronic responses to exercise. Redox signalling pathways involved in adaptive responses in skeletal muscle to exercise are not clearly elucidated at present, and further research is required to better define important signalling pathways involved. Evidence of beneficial or detrimental effects of specific antioxidant compounds on exercise adaptations in muscle is similarly limited, particularly in human subjects. Future research is required to not only investigate effects of specific antioxidant compounds on skeletal muscle exercise adaptations, but also to better establish mechanisms of action of specific antioxidants in vivo. Although we feel it remains somewhat premature to make clear recommendations in relation to application of specific antioxidant compounds in different exercise settings, a bulk of evidence suggests that N-acetylcysteine (NAC) is ergogenic through its effects on maintenance of muscle force production during sustained fatiguing events. Nevertheless, a current lack of evidence from studies using performance tests representative of athletic competition and a potential for adverse effects with high doses (>70mg/kg body mass) warrants caution in its use for performance enhancement. In addition, evidence implicates high dose vitamin C (1g/day) and E

Rewiring of cellular membrane homeostasis by picornaviruses.

PubMed

Belov, George A; Sztul, Elizabeth

2014-09-01

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Olanzapine and aripiprazole differentially affect glucose uptake and energy metabolism in human mononuclear blood cells.

PubMed

Stapel, Britta; Kotsiari, Alexandra; Scherr, Michaela; Hilfiker-Kleiner, Denise; Bleich, Stefan; Frieling, Helge; Kahl, Kai G

2017-05-01

The use of antipsychotics carries the risk of metabolic side effects, such as weight gain and new onset type-2 diabetes mellitus. The mechanisms of the observed metabolic alterations are not fully understood. We compared the effects of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (aripiprazole), on glucose metabolism. Primary human peripheral blood mononuclear cells (PBMC) were isolated and stimulated with olanzapine or aripiprazole for 72 h. Cellular glucose uptake was analyzed in vitro by 18F-FDG uptake. Further measurements comprised mRNA expression of glucose transporter (GLUT) 1 and 3, GLUT1 protein expression, DNA methylation of GLUT1 promoter region, and proteins involved in downstream glucometabolic processes. We observed a 2-fold increase in glucose uptake after stimulation with aripiprazole. In contrast, olanzapine stimulation decreased glucose uptake by 40%, accompanied by downregulation of the cellular energy sensor AMP activated protein kinase (AMPK). GLUT1 protein expression increased, GLUT1 mRNA expression decreased, and GLUT1 promoter was hypermethylated with both antipsychotics. Pyruvat-dehydrogenase (PDH) complex activity decreased with olanzapine only. Our findings suggest that the atypical antipsychotics olanzapine and aripiprazole differentially affect energy metabolism in PBMC. The observed decrease in glucose uptake in olanzapine stimulated PBMC, accompanied by decreased PDH point to a worsening in cellular energy metabolism not compensated by AMKP upregulation. In contrast, aripiprazole stimulation lead to increased glucose uptake, while not affecting PDH complex expression. The observed differences may be involved in the different metabolic profiles observed in aripiprazole and olanzapine treated patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative evaluation of nano-CuO crossing Caco-2 cell monolayers and cellular uptake

NASA Astrophysics Data System (ADS)

Chen, Gao; Lianqin, Zhu; Fenghua, Zhu; Fang, Zheng; Mingming, Song; Kai, Huang

2015-04-01

Different concentrations of CuSO4, micro-CuO, and nano-CuO were added to Caco-2 cell monolayers to study the absorption and transport characteristics in this epithelial cell model. Nano-CuO nanoparticles had a diameter of 10-20 nm. Inhibitors of endocytosis were used to explore whether nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and to ascertain the endocytotic pathway that is involved in the transport process. The apparent permeability coefficient ( P app) of CuSO4 and nano-CuO increased with the Cu concentration in the culture medium ( p < 0.05). The micro-CuO of different concentrations had no significant impact on the P app value of Caco-2 cells ( p > 0.05). When the Cu concentration in the culture medium was in the range 31.25-500 μM, the P app value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO4. The latter was also significantly higher than that when cells were incubated with micro-CuO ( p < 0.05). The amount of Cu transport increased with the increase of CuSO4 concentration in the culture medium. After 90 min, the amount of transport began to saturate, and the transport rate of Cu declined with the increase of CuSO4 concentration. For the cells incubated with nano-CuO, the amount of Cu transport increased with the increase of nano-CuO concentration, but did not show an obvious saturation with the extension of transport time. Nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and were found in the cytoplasm, vesicles, lysosomes, and cell nuclei. Several inhibitors of endocytosis effectively prevented the entry of nano-CuO into the Caco-2 cells. It was concluded that nano-CuO particles can enter the Caco-2 cells through several cellular endocytotic pathways.

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.

PubMed

Becker, Björn; Schnöder, Tina; Schmitt, Manfred J

2016-12-06

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast.

Effects of calcium on hepatocyte iron uptake from transferrin, iron-pyrophosphate and iron-ascorbate.

PubMed

Nilsen, T

1991-10-16

Calcium stimulates hepatocyte iron uptake from transferrin, ferric-iron-pyrophosphate and ferrous-iron-ascorbate. Maximal stimulation of iron uptake is observed at 1-1.5 mM of extra-cellular calcium and the effect is reversible and immediate. Neither the receptor affinity for transferrin, nor the total amounts of transferrin associated with the cells or the rate of transferrin endocytosis are significantly affected by calcium. In the presence of calcium the rate of iron uptake of non-transferrin bound iron increases abruptly at approximate 17 degrees C and 27 degrees C and as assessed by Arrhenius plots, the activation energy is reduced in a calcium dependent manner at approx. 27 degrees C. At a similar temperature, i.e., between 25 degrees C and 28 degrees C, calcium increases the rates of cellular iron uptake from transferrin in a way that is not reflected in the rate of transferrin endocytosis. By the results of this study it is concluded that calcium increases iron transport across the plasma membrane by a mechanism dependent on membrane fluidity.

Magnetic nanoparticles to recover cellular organelles and study the time resolved nanoparticle-cell interactome throughout uptake.

PubMed

Bertoli, Filippo; Davies, Gemma-Louise; Monopoli, Marco P; Moloney, Micheal; Gun'ko, Yurii K; Salvati, Anna; Dawson, Kenneth A

2014-08-27

Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the 'hard corona' that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dual-drug delivery by porous silicon nanoparticles for improved cellular uptake, sustained release, and combination therapy.

PubMed

Wang, Chang-Fang; Mäkilä, Ermei M; Kaasalainen, Martti H; Hagström, Marja V; Salonen, Jarno J; Hirvonen, Jouni T; Santos, Hélder A

2015-04-01

Dual-drug delivery of antiangiogenic and chemotherapeutic drugs can enhance the therapeutic effect for cancer therapy. Conjugation of methotrexate (MTX) to porous silicon (PSi) nanoparticles (MTX-PSi) with positively charged surface can improve the cellular uptake of MTX and inhibit the proliferation of cancer cells. Herein, MTX-PSi conjugates sustained the release of MTX up to 96 h, and the released fragments including MTX were confirmed by mass spectrometry. The intracellular distribution of the MTX-PSi nanoparticles was confirmed by transmission electron microscopy. Compared to pure MTX, the MTX-PSi achieved similar inhibition of cell proliferation in folate receptor (FR) over-expressing U87 MG cancer cells, and a higher effect in low FR-expressing EA.hy926 cells. Nuclear fragmentation analysis demonstrated programmed cell apoptosis of MTX-PSi in the high/low FR-expressing cancer cells, whereas PSi alone at the same dose had a minor effect on cell apoptosis. Finally, the porous structure of MTX-PSi enabled a successful concomitant loading of another anti-angiogenic hydrophobic drug, sorafenib, and considerably enhanced the dissolution rate of sorafenib. Overall, the MTX-PSi nanoparticles can be used as a platform for combination chemotherapy by simultaneously enhancing the dissolution rate of a hydrophobic drug and sustaining the release of a conjugated chemotherapeutic drug. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cellular proteostasis: degradation of misfolded proteins by lysosomes

PubMed Central

Jackson, Matthew P.

2016-01-01

Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

Global functional analyses of cellular responses to pore-forming toxins.

PubMed

Kao, Cheng-Yuan; Los, Ferdinand C O; Huffman, Danielle L; Wachi, Shinichiro; Kloft, Nicole; Husmann, Matthias; Karabrahimi, Valbona; Schwartz, Jean-Louis; Bellier, Audrey; Ha, Christine; Sagong, Youn; Fan, Hui; Ghosh, Partho; Hsieh, Mindy; Hsu, Chih-Shen; Chen, Li; Aroian, Raffi V

2011-03-01

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Characterization of nanoparticle uptake by endothelial cells.

PubMed

Davda, Jasmine; Labhasetwar, Vinod

2002-02-21

Endothelium is an important target for drug or gene therapy because of its important role in the biological system. In this paper, we have characterized nanoparticle uptake by endothelial cells in cell culture. Nanoparticles were formulated using poly DL-lactide-co-glycolide polymer containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker. It was observed that the cellular uptake of nanoparticles depends on the time of incubation and the concentration of nanoparticles in the medium. The uptake of nanoparticles was rapid with confocal microscopy demonstrating their localization mostly in the cytoplasm. The mitogenic study demonstrated biocompatability of nanoparticles with the cells. The study thus demonstrates that nanoparticles could be used for localizing therapeutic agents or gene into endothelial cells. Nanoparticles localized in the endothelium could provide prolonged drug effects because of their sustained release characterics, and also could protect the encapsulated agent from enzymatic degradation.

Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery

NASA Astrophysics Data System (ADS)

Terracciano, Monica; Shahbazi, Mohammad-Ali; Correia, Alexandra; Rea, Ilaria; Lamberti, Annalisa; de Stefano, Luca; Santos, Hélder A.

2015-11-01

Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 μg mL-1 after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 μg mL-1 and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles.Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles

SM22{alpha}-induced activation of p16{sup INK4a}/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of {gamma}-radiation and doxorubicin in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Tae Rim; Lee, Hee Min; Lee, So Yong

Research highlights: {yields} SM22{alpha} overexpression in HepG2 cells leads cells to a growth arrest state, and the treatment of a subclinical dose of {gamma}-radiation or doxorubicin promotes cellular senescence. {yields} SM22{alpha} overexpression elevates p16{sup INK4a} followed by pRB activation, but there are no effects on p53/p21{sup WAF1/Cip1} pathway. {yields} SM22{alpha}-induced MT-1G activates p16{sup INK4a}/pRB pathway, which promotes cellular senescence by damaging agents. -- Abstract: Smooth muscle protein 22-alpha (SM22{alpha}) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22{alpha} overexpression confersmore » resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22{alpha} overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of {gamma}-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 {mu}g/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21{sup WAF1/Cip1} induction or p16{sup INK4a}/retinoblastoma protein (pRB) activation. SM22{alpha} overexpression in HepG2 cells elevated p16{sup INK4a} followed by pRB activation, but did not activate the p53/p21{sup WAF1/Cip1} pathway. Moreover, MT-1G, which is induced by SM22{alpha} overexpression, was involved in the activation of the p16{sup INK4a}/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22{alpha} modulates cellular senescence caused by damaging agents via regulation of the p16{sup INK4a}/pRB pathway in HepG2 cells and that these effects of SM22{alpha} are partially mediated by MT-1G.« less

Cellular cytotoxic response induced by highly purified multi-wall carbon nanotube in human lung cells.

PubMed

Tsukahara, Tamotsu; Haniu, Hisao

2011-06-01

Carbon nanotubes, a promising nanomaterial with unique characteristics, have applications in a variety of fields. The cytotoxic effects of carbon nanotubes are partially due to the induction of oxidative stress; however, the detailed mechanisms of nanotube cytotoxicity and their interaction with cells remain unclear. In this study, the authors focus on the acute toxicity of vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) by high-temperature thermal treatment. The authors exposed human bronchial epithelial cells (BEAS-2B) to HTT2800 and measured the cellular uptake, mitochondrial function, cellular LDH release, apoptotic signaling, reactive oxygen species (ROS) generation and pro-inflammatory cytokine release. The HTT2800-exposed cells showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. However, the exposed cells showed no obvious intracellular ROS generation. These cellular and molecular findings suggest that HTT2800 could cause a potentially adverse inflammatory response in BEAS-2B cells.

Modeling the mechanics of cancer: effect of changes in cellular and extra-cellular mechanical properties.

PubMed

Katira, Parag; Bonnecaze, Roger T; Zaman, Muhammad H

2013-01-01

Malignant transformation, though primarily driven by genetic mutations in cells, is also accompanied by specific changes in cellular and extra-cellular mechanical properties such as stiffness and adhesivity. As the transformed cells grow into tumors, they interact with their surroundings via physical contacts and the application of forces. These forces can lead to changes in the mechanical regulation of cell fate based on the mechanical properties of the cells and their surrounding environment. A comprehensive understanding of cancer progression requires the study of how specific changes in mechanical properties influences collective cell behavior during tumor growth and metastasis. Here we review some key results from computational models describing the effect of changes in cellular and extra-cellular mechanical properties and identify mechanistic pathways for cancer progression that can be targeted for the prediction, treatment, and prevention of cancer.

Controlling Cellular Endocytosis at the Nanoscale

NASA Astrophysics Data System (ADS)

Battaglia, Giuseppe

2011-03-01

, amphiphilic molecules, and hydrophilic molecules without affecting the viability of cells or even triggering inflammatory pathways. Finally we show how size, surface chemistry and surface topology of the vesicles affect their interaction with the cell membrane and hence their cellular uptake. References: C. Lo Presti, M. Massignani, T. Smart, H. Lomas, and G. Battaglia J. Mater. Chem. (2009) 19, 3576-3590 H. Lomas, I. Canton, S. MacNeil, J. Du, S.P. Armes, A.J. Ryan, A.L. Lewis and G. Battaglia Adv. Mater. (2007). 19, 4238-4243 M. Massignani, I. Canton, N. Patikarnmonthon, N. J. Warren, S. P. Armes, A. L. Lewis and G. Battaglia, Nature Prec., 2010, http://hdl.handle.net/10101/npre.2010.4427.1 M. Massignani, C. LoPresti, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis and G. Battaglia Small, 2009, 5, 2424-2432. M. Massignani, T. Sun, A. Blanazs, V. Hearnden, I. Canton, P. Desphande, S. Armes, S. MacNeil, A. Lewis and G. Battaglia PLoS One, 2010, 5, e10459.

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake

PubMed Central

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-01-01

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1. PMID:26083118

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake.

PubMed

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-06-15

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.

PubMed

Russell, R R; Bergeron, R; Shulman, G I; Young, L H

1999-08-01

Insulin increases glucose uptake through the translocation of GLUT-4 via a pathway mediated by phosphatidylinositol 3-kinase (PI3K). In contrast, myocardial glucose uptake during ischemia and hypoxia is stimulated by the translocation of GLUT-4 to the surface of cardiac myocytes through a PI3K-independent pathway that has not been characterized. AMP-activated protein kinase (AMPK) activity is also increased by myocardial ischemia, and we examined whether AMPK stimulates glucose uptake and GLUT-4 translocation. In isolated rat ventricular papillary muscles, 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, as well as cyanide-induced chemical hypoxia and insulin, increased 2-[(3)H]deoxyglucose uptake two- to threefold. Wortmannin, a PI3K inhibitor, did not affect either the AICAR- or the cyanide-stimulated increase in deoxyglucose uptake but eliminated the insulin-stimulated increase in deoxyglucose uptake. Immunofluorescence studies demonstrated translocation of GLUT-4 to the myocyte sarcolemma in response to stimulation with AICAR, cyanide, or insulin. Preincubation of papillary muscles with the kinase inhibitor iodotubercidin or adenine 9-beta-D-arabinofuranoside (araA), a precursor of araATP (a competitive inhibitor of AMPK), decreased AICAR- and cyanide-stimulated glucose uptake but did not affect basal or insulin-stimulated glucose uptake. In vivo infusion of AICAR caused myocardial AMPK activation and GLUT-4 translocation in the rat. We conclude that AMPK activation increases cardiac muscle glucose uptake through translocation of GLUT-4 via a pathway that is independent of PI3K. These findings suggest that AMPK activation may be important in ischemia-induced translocation of GLUT-4 in the heart.

The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation.

PubMed

Santos, J M; Benite-Ribeiro, S A; Queiroz, G; Duarte, J A

2014-12-01

Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3-kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin-activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho-aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho-aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle. Copyright © 2014 John Wiley & Sons, Ltd.

AMPK and Exercise: Glucose Uptake and Insulin Sensitivity

PubMed Central

2013-01-01

AMPK is an evolutionary conserved sensor of cellular energy status that is activated during exercise. Pharmacological activation of AMPK promotes glucose uptake, fatty acid oxidation, mitochondrial biogenesis, and insulin sensitivity; processes that are reduced in obesity and contribute to the development of insulin resistance. AMPK deficient mouse models have been used to provide direct genetic evidence either supporting or refuting a role for AMPK in regulating these processes. Exercise promotes glucose uptake by an insulin dependent mechanism involving AMPK. Exercise is important for improving insulin sensitivity; however, it is not known if AMPK is required for these improvements. Understanding how these metabolic processes are regulated is important for the development of new strategies that target obesity-induced insulin resistance. This review will discuss the involvement of AMPK in regulating skeletal muscle metabolism (glucose uptake, glycogen synthesis, and insulin sensitivity). PMID:23441028

Evaluation of cellular uptake and gene transfer efficiency of pegylated poly-L-lysine compacted DNA: implications for cancer gene therapy.

PubMed

Walsh, M; Tangney, M; O'Neill, M J; Larkin, J O; Soden, D M; McKenna, S L; Darcy, R; O'Sullivan, G C; O'Driscoll, C M

2006-01-01

Recent success in phase I/II clinical trials (Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; Oette, S. M.; Payne, J. M.; Muhammad, O.; Ziady, A. G.; Moen, R. C.; Cooper, M. J. Hum. Gene Ther. 2004, 15 (12), 1255-69) has highlighted pegylated poly-L-lysine (C1K30-PEG) as a nonviral gene delivery agent capable of achieving clinically significant gene transfer levels in vivo. This study investigates the potential of a C1K30-PEG gene delivery system for cancer gene therapy and evaluates its mode of cellular entry with the purpose of developing an optimally formulated prototype for tumor cell transfection. C1K30-PEG complexes have a neutral charge and form rod-like and toroid-like nanoparticles. Comparison of the transfection efficiency achieved by C1K30-PEG with other cationic lipid and polymeric vectors demonstrates that C1K30-PEG transfects cells more efficiently than unpegylated poly-L-lysine and compares well to commercially available vectors. In vivo gene delivery by C1K30-PEG nanoparticles to a growing subcutaneous murine tumor was also demonstrated. To determine potential barriers to C1K30-PEG gene delivery, the entry mechanism and intracellular fate of rhodamine labeled complexes were investigated. Using cellular markers to delineate the pathway taken by the complexes upon cellular entry, only minor colocalization was observed with EEA-1, a marker of early endosomes. No colocalization was observed between the complexes and the transferrin receptor, which is a marker for clathrin-coated pits. In addition, complexes were not observed to enter late endosomes/lysosomes. Cellular entry of the complexes was completely inhibited by the macropinocytosis inhibitor, amiloride, indicating that the complexes enter cells via macropinosomes. Such mechanistic studies are an essential step to support future rational design of pegylated poly-L-lysine vectors to improve the

Effect of Thiols, Zinc, and Redox Conditions on Hg Uptake in Shewanella oneidensis

DOE PAGES

Szczuka, Aleksandra; Morel, Francois M. M.; Schaefer, Jeffra K.

2015-05-18

Mercury uptake in bacteria represents a key first step in the production and accumulation Of methylmercury in biota. Previous experiments with mercury methylating bacteria have shown that Hg uptake is enhanced by some thiols, in particular cysteine, and that it is an energy-dependent process through heavy Metal TA transporters. In this study, we examine Hg uptake in the nonmethylating facultative aerobe, Shewanella oneidensis, under both anaerobic and aerobic conditions. Our results demonstrate similar characteristics of the Hg uptake system to those of the Hg methylating strains: uptake is enhanced in the presence of some thiols but not others; uptake ismore » energy dependent as evidenced by inhibition by a protonophore; and uptake is inhibited by high Zn(II) concentrations. Initial cellular uptake rates in S. oneidensis were remarkably similar under aerobic and fumarate-reducing conditions. In conclusion, these data support a similar Hg(II) uptake mechanism within the proteobacteria of accidental Hg(II) transport through heavy metal transporters with similar rates of uptake but differences in the ability to take up Hg bound to different thiols.« less

Uptake of a fluorescent L-glucose derivative 2-NBDLG into three-dimensionally accumulating insulinoma cells in a phloretin-sensitive manner.

PubMed

Sasaki, Ayako; Nagatomo, Katsuhiro; Ono, Koki; Yamamoto, Toshihiro; Otsuka, Yuji; Teshima, Tadashi; Yamada, Katsuya

2016-01-01

Of two stereoisomers of glucose, only D- and not L-glucose is abundantly found in nature, being utilized as an essential fuel by most organisms. The uptake of D-glucose into mammalian cells occurs through glucose transporters such as GLUTs, and this process has been effectively monitored by a fluorescent D-glucose derivative 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) at the single cell level. However, since fluorescence is an arbitrary measure, we have developed a fluorescent analog of L-glucose 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), as a negative control substrate for more accurately identifying the stereoselectivity of the uptake. Interestingly, a small portion of mouse insulinoma cells MIN6 abundantly took up 2-NBDLG at a late culture stage (≳ 10 days in vitro, DIV) when multi-cellular spheroids exhibiting heterogeneous nuclei were formed, whereas no such uptake was detected at an early culture stage (≲ 6 DIV). The 2-NBDLG uptake was persistently observed in the presence of a GLUT inhibitor cytochalasin B. Neither D- nor L-glucose in 50 mM abolished the uptake. No significant inhibition was detected by inactivating sodium/glucose cotransporters (SGLTs) with Na(+)-free condition. To our surprise, the 2-NBDLG uptake was totally inhibited by phloretin, a broad spectrum inhibitor against transporters/channels including GLUTs and aquaporins. From these, a question might be raised if non-GLUT/non-SGLT pathways participate in the 2-NBDLG uptake into spheroid-forming MIN6 insulinoma. It might also be worthwhile investigating whether 2-NBDLG can be used as a functional probe for detecting cancer, since the nuclear heterogeneity is among critical features of malignancy.

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

PubMed

Yun, Bo; Azad, Mohammad A K; Nowell, Cameron J; Nation, Roger L; Thompson, Philip E; Roberts, Kade D; Velkov, Tony; Li, Jian

2015-12-01

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Label-Free Raman Microspectral Analysis for Comparison of Cellular Uptake and Distribution between Non-Targeted and EGFR-Targeted Biodegradable Polymeric Nanoparticles

PubMed Central

Chernenko, Tatyana; Buyukozturk, Fulden; Miljkovic, Milos; Carrier, Rebecca; Diem, Max; Amiji, Mansoor

2013-01-01

Active targeted delivery of nanoparticle-encapsulated agents to tumor cells in vivo is expected to enhance therapeutic effect with significantly less non-specific toxicity. Active targeting is based on surface modification of nanoparticles with ligands that bind with extracellular targets and enhance payload delivery in the cells. In this study, we have used label-free Raman micro-spectral analysis and kinetic modeling to study cellular interactions and intracellular delivery of C6-ceramide using a non-targeted and an epidermal growth factor receptor (EGFR) targeted biodegradable polymeric nano-delivery systems, in EGFR-expressing human ovarian adenocarcinoma (SKOV3) cells. The results show that EGFR peptide-modified nanoparticles were rapidly internalized in SKOV3 cells leading to significant intracellular accumulation as compared to non-specific uptake by the non-targeted nanoparticles. Raman micro-spectral analysis enables visualization and quantification of the carrier system, drug-load, and responses of the biological systems interrogated, without exogenous staining and labeling procedures. PMID:24298430

Vhl deletion in osteoblasts boosts cellular glycolysis and improves global glucose metabolism

PubMed Central

Dirckx, Naomi; Tower, Robert J.; Mercken, Evi M.; Moreau-Triby, Caroline; Breugelmans, Tom; Nefyodova, Elena; Cardoen, Ruben; Mathieu, Chantal; Van der Schueren, Bart; Confavreux, Cyrille B.; Clemens, Thomas L.

2018-01-01

The skeleton has emerged as an important regulator of systemic glucose homeostasis, with osteocalcin and insulin representing prime mediators of the interplay between bone and energy metabolism. However, genetic evidence indicates that osteoblasts can influence global energy metabolism through additional, as yet unknown, mechanisms. Here, we report that constitutive or postnatally induced deletion of the hypoxia signaling pathway component von Hippel–Lindau (VHL) in skeletal osteolineage cells of mice led to high bone mass as well as hypoglycemia and increased glucose tolerance, not accounted for by osteocalcin or insulin. In vitro and in vivo data indicated that Vhl-deficient osteoblasts displayed massively increased glucose uptake and glycolysis associated with upregulated HIF-target gene expression, resembling the Warburg effect that typifies cancer cells. Overall, the glucose consumption by the skeleton was increased in the mutant mice, as revealed by 18F-FDG radioactive tracer experiments. Moreover, the glycemia levels correlated inversely with the level of skeletal glucose uptake, and pharmacological treatment with the glycolysis inhibitor dichloroacetate (DCA), which restored glucose metabolism in Vhl-deficient osteogenic cells in vitro, prevented the development of the systemic metabolic phenotype in the mutant mice. Altogether, these findings reveal a novel link between cellular glucose metabolism in osteoblasts and whole-body glucose homeostasis, controlled by local hypoxia signaling in the skeleton. PMID:29431735

Cellular compartmentalization of secondary metabolism

PubMed Central

Kistler, H. Corby; Broz, Karen

2015-01-01

Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g., amino acids, acetyl CoA, NADPH), enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported. PMID:25709603

Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.