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Sample records for generates intracellular multilamellar

  1. Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation

    PubMed Central

    Matias, Andreza Cândido; Manieri, Tânia Maria; Cerchiaro, Giselle

    2016-01-01

    We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation. PMID:27123155

  2. Autoantibodies to intracellular antigens: generation and pathogenetic role.

    PubMed

    Racanelli, Vito; Prete, Marcella; Musaraj, Gerta; Dammacco, Franco; Perosa, Federico

    2011-06-01

    Autoantibodies to intracellular antigens form a large family of immunoglobulins directed to a variety of ubiquitously expressed intracellular molecules, including numerous enzymes, some ribonucleoproteins and double-stranded DNA. These anti-self antibodies have been found to be selectively expressed in sera of patients with several systemic (non-organ-specific) autoimmune diseases, such as systemic sclerosis (SSc), SLE, mixed connective tissue disease, Sjögren's syndrome and idiopathic myopathies. Despite their important diagnostic and prognostic value and their utility in assessing disease activity, little is known about the molecular mechanisms involved in their generation and role in autoimmune diseases nor is it known why particular autoantibodies are preferentially expressed in certain diseases. Here, we review the different lines of research which are presently being conducted to understand how these autoantibodies are generated (e.g. through apoptotic body formation, molecular mimicry and other mechanisms) and how they encounter antigen in order to cause an autoimmune disease. The recently reported mechanism of intracellular immunity mediated by Ro52 (or tripartite motif containing 21, TRIM21) in a cellular model of adenovirus infection is opening new perspectives for studying the effects of autoantibodies once they get inside cells. PMID:21397735

  3. Biological Hydrogels Formed by Swollen Multilamellar Liposomes.

    PubMed

    Cheng, Chih-Yang; Wang, Ting-Yu; Tung, Shih-Huang

    2015-12-15

    The self-assembly of lecithin-bile salt mixtures in solutions has long been an important research topic, not only because they are both biosurfactants closely relevant to physiological functions but also for the potential biomedical applications. In this paper, we report an unusual biological hydrogel formed by mixing bile salts and lecithin at low bile salt/lecithin molar ratios (B0) in water. The gel can be prepared at a total lipid concentration as low as ∼15 wt %, and the solidlike property of the solutions was confirmed by dynamic rheological measurements. We used cryo-TEM and SAXS/SANS techniques to probe the self-assembled structure and clearly evidence that the gel is made up of jammed swollen multilamellar vesicles (liposomes), instead of typical fibrous networks found in conventional gels. A mechanism-based on the strong repulsion between bilayers due to the incorporation of negatively charged bile salts is proposed to explain the swelling of the liposomes. In addition to gel, a series of phases, including viscoelastic, gel-like, and low-viscosity fluids, can be created by increasing B0. Such a variety of phase behaviors are caused by the transformation of bilayers into cylindrical and spheroidal micelles upon the change of the effective molecular geometry with B0. PMID:26574777

  4. Light generation of intracellular Ca2+ signals by a genetically encoded protein BACCS

    PubMed Central

    Ishii, Tomohiro; Sato, Koji; Kakumoto, Toshiyuki; Miura, Shigenori; Touhara, Kazushige; Takeuchi, Shoji; Nakata, Takao

    2015-01-01

    Ca2+ signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca2+ channel switch (BACCS), as an optogenetic tool for generating Ca2+ signals. BACCS opens Ca2+-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca2+ concentration more rapidly, such that Ca2+ elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca2+. Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca2+ signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies. PMID:26282514

  5. Intracellular bottom-up generation of targeted nanosensors for single-molecule imaging

    NASA Astrophysics Data System (ADS)

    Hou, Yanyan; Arai, Satoshi; Kitaguchi, Tetsuya; Suzuki, Madoka

    2016-02-01

    Organic dyes are useful tools for sensing cellular activities but unfavorable in single-molecule imaging, whereas quantum dots (QDs) are widely applied in single-molecule imaging but with few sensing applications. Here, to visualize cellular activities by monitoring the response of a single probe in living cells, we propose a bottom-up approach to generate nanoprobes where four organic dyes are conjugated to tetravalent single-chain avidin (scAVD) proteins via an intracellular click reaction. We demonstrate that the nanoprobes, exhibiting increased brightness and enhanced photostability, were detectable as single dots in living cells. The ease of intracellular targeting allowed the tracking of endoplasmic reticulum (ER) remodeling with nanometer spatial resolution. Conjugating thermosensitive dyes generated temperature-sensitive nanoprobes on ER membranes that successfully monitored local temperature changes in response to external heat pulses. Our approach is potentially a suitable tool for visualizing localized cellular activities with single probe sensitivity in living cells.Organic dyes are useful tools for sensing cellular activities but unfavorable in single-molecule imaging, whereas quantum dots (QDs) are widely applied in single-molecule imaging but with few sensing applications. Here, to visualize cellular activities by monitoring the response of a single probe in living cells, we propose a bottom-up approach to generate nanoprobes where four organic dyes are conjugated to tetravalent single-chain avidin (scAVD) proteins via an intracellular click reaction. We demonstrate that the nanoprobes, exhibiting increased brightness and enhanced photostability, were detectable as single dots in living cells. The ease of intracellular targeting allowed the tracking of endoplasmic reticulum (ER) remodeling with nanometer spatial resolution. Conjugating thermosensitive dyes generated temperature-sensitive nanoprobes on ER membranes that successfully monitored local

  6. Ectodomain Shedding of Interleukin-2 Receptor β and Generation of an Intracellular Functional Fragment*

    PubMed Central

    de Oca B., Pavel Montes; Malardé, Valerie; Proust, Richard; Dautry-Varsat, Alice; Gesbert, Franck

    2010-01-01

    Interleukin-2 (IL-2) regulates different functions of various lymphoid cell subsets. These are mediated by its binding to the IL-2 receptor (IL-2R) composed of three subunits (IL2-Rα, -β, and -γc). IL-2Rβ is responsible for the activation of several signaling pathways. Ectodomain shedding of membrane receptors is thought to be an important mechanism for down-regulation of cell surface receptor abundance but is also emerging as a mechanism that cell membrane-associated molecules require for proper action in vivo. Here, we demonstrate that IL-2Rβ is cleaved in cell lines of different origin, including T cells, generating an intracellular 37-kDa fragment (37βic) that comprises the full intracellular C-terminal and transmembrane domains. Ectodomain shedding of IL-2Rβ decreases in a mutant deleted of the juxtamembrane region, where cleavage is predicted to occur, and is inhibited by tissue inhibitor of metalloproteases-3. 37βic is tyrosine-phosphorylated and associates with STAT-5, a canonic signal transducer of IL-2R. Finally, lymphoid cell transfection with a truncated form of IL-2Rβ mimicking 37βic increases their proliferation. These data indicate that IL-2Rβ is subject to ectodomain shedding generating an intracellular fragment biologically functional, because (i) it is phosphorylated, (ii) it associates with STAT5A, and (iii) it increases cell proliferation. PMID:20495002

  7. Multilamellar liposomes entrapping aminosilane-modified maghemite nanoparticles: "magnetonions".

    PubMed

    Meyre, Marie-Edith; Clérac, Rodolphe; Mornet, Stéphane; Duguet, Etienne; Dole, François; Nallet, Frédéric; Lambert, Olivier; Trépout, Sylvain; Faure, Chrystel

    2010-10-21

    4.6 nm-sized aminosilane-modified maghemite (γ-Fe(2)O(3)) nanoparticles (aMNPs) were synthesized and encapsulated into onion-type multilamellar vesicles of soybean phosphatidylcholine (90%mol) and monoolein (10%mol). The magnetic multilamellar vesicles were obtained by shearing lipids with an aqueous dispersion of the preformed aMNPs (ferrofluid). The influence of ferrofluid concentration on the stability of the constitutive lamellar phase and the resulting dispersed onions was analyzed by small-angle X-ray diffraction (SAXD) and cryo-TEM imaging, respectively. When [Fe(III)] <60 mM, stable, magnetic onions were produced with aMNPs inserted inside onion water compartments as isolated or aggregated particles. Encapsulation efficiencies were measured by EPR spectroscopy and magnetic measurements: much higher values (up to 75%) than unilamellar liposomes were found. The deduced aMNP-to-onion ratio increased with ferrofluid concentration before reaching a maximal value of ca. 45 as confirmed by cryo-TEM imaging. When [Fe(III)] >60 mM, uni- or oligolamellar vesicles in addition to onions formed, probably because of a two-phase separation between an aMNP-rich phase and an aMNP-containing lamellar phase as revealed by SAXD. PMID:20820553

  8. Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles

    NASA Astrophysics Data System (ADS)

    Lavado, Andrea S.; Chauhan, Veeren M.; Alhaj Zen, Amer; Giuntini, Francesca; Jones, D. Rhodri E.; Boyle, Ross W.; Beeby, Andrew; Chan, Weng C.; Aylott, Jonathan W.

    2015-08-01

    Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn

  9. Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells.

    PubMed

    Mainz, Emilie R; Dobes, Nicholas C; Allbritton, Nancy L

    2015-08-01

    The ability to track intracellular peptide proteolysis at the single cell level is of growing interest, particularly as short peptide sequences continue to play important roles as biosensors, therapeutics, and endogenous participants in antigen processing and intracellular signaling. We describe a rapid and inexpensive methodology to generate fluorescent peptide fragments from a parent sequence with diverse chemical properties, including aliphatic, nonpolar, basic, acidic, and non-native amino acids. Four peptide sequences with existing biochemical applications were fragmented using incubation with Pronase E and/or formic acid, and in each case a complete set of fluorescent fragments was generated for use as proteolysis standards in chemical cytometry. Fragment formation and identity was monitored with capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-MS) to confirm the presence of all sequences and yield fragmentation profiles across Pronase E concentrations which can readily be used by others. As a pilot study, Pronase E-generated standards from an Abl kinase sensor and an ovalbumin antigenic peptide were then employed to identify proteolysis products arising from the metabolism of these sequences in single cells. The Abl kinase sensor fragmented at 4.2 ± 4.8 zmol μM(-1) s(-1) and the majority of cells possessed similar fragment identities. In contrast, an ovalbumin epitope peptide was degraded at 8.9 ± 0.1 zmol μM(-1) s(-1), but with differential fragment formation between individual cells. Overall, Pronase E-generated peptide standards were a rapid and efficient method to identify proteolysis products from cells. PMID:26171808

  10. Shear-Induced Deformation of Surfactant Multilamellar Vesicles

    NASA Astrophysics Data System (ADS)

    Pommella, Angelo; Caserta, Sergio; Guida, Vincenzo; Guido, Stefano

    2012-03-01

    Surfactant multilamellar vesicles (SMLVs) play a key role in the formulation of many industrial products, such as detergents, foodstuff, and cosmetics. In this Letter, we present the first quantitative investigation of the flow behavior of single SMLVs in a shearing parallel plate apparatus. We found that SMLVs are deformed and oriented by the action of shear flow while keeping constant volume and exhibit complex dynamic modes (i.e., tumbling, breathing, and tank treading). This behavior can be explained in terms of an excess area (as compared to a sphere of the same volume) and of microstructural defects, which were observed by 3D shape reconstruction through confocal microscopy. Furthermore, the deformation and orientation of SMLVs scale with radius R in analogy with emulsion droplets and elastic capsules (instead of R3, such as in unilamellar vesicles). A possible application of the physical insight provided by this Letter is in the rationale design of processing methods of surfactant-based systems.

  11. How aluminum, an intracellular ROS generator promotes hepatic and neurological diseases: the metabolic tale.

    PubMed

    Han, Sungwon; Lemire, Joseph; Appanna, Varun P; Auger, Christopher; Castonguay, Zachary; Appanna, Vasu D

    2013-04-01

    Metal pollutants are a global health risk due to their ability to contribute to a variety of diseases. Aluminum (Al), a ubiquitous environmental contaminant is implicated in anemia, osteomalacia, hepatic disorder, and neurological disorder. In this review, we outline how this intracellular generator of reactive oxygen species (ROS) triggers a metabolic shift towards lipogenesis in astrocytes and hepatocytes. This Al-evoked phenomenon is coupled to diminished mitochondrial activity, anerobiosis, and the channeling of α-ketoacids towards anti-oxidant defense. The resulting metabolic reconfiguration leads to fat accumulation and a reduction in ATP synthesis, characteristics that are common to numerous medical disorders. Hence, the ability of Al toxicity to create an oxidative environment promotes dysfunctional metabolic processes in astrocytes and hepatocytes. These molecular events triggered by Al-induced ROS production are the potential mediators of brain and liver disorders. PMID:23463459

  12. Kinetics of a Multilamellar Lipid Vesicle Ripening: Simulation and Theory.

    PubMed

    Xu, Rui; He, Xuehao

    2016-03-10

    Lipid vesicle ripening via unimolecular diffusion and exchange greatly influences the evolution of complex vesicle structure. However, this behavior is difficult to capture using conventional experimental technology and molecular simulation. In the present work, the ripening of a multilamellar lipid vesicle (MLV) is effectively explored using a mesoscale coarse-grained molecular model. The simulation reveals that a small MLV evolves into a unilamellar vesicle over a very long time period. In this process, only the outermost bilayer inflates, and the inner bilayers shrink. With increasing MLV size, the ripening process becomes complex and depends on competition between a series of adjacent bilayers in the MLV. To understand the diffusion behavior of the unimolecule, the potentials of mean force (PMFs) of a single lipid molecule across unilamellar vesicles with different sizes are calculated. It is found that the PMF of lipid dissociation from the inner layer is different than that of the outer layer, and the dissociation energy barrier sensitively depends on the curvature of the bilayer. A kinetics theoretical model of MLV ripening that considers the lipid dissociation energy for curved bilayers is proposed. The model successfully interprets the MLV ripening process with various numbers of bilayers and shows potential to predict the ripening kinetics of complex lipid vesicles. PMID:26882997

  13. Crosslinked multilamellar liposomes for controlled delivery of anticancer drugs.

    PubMed

    Joo, Kye-Il; Xiao, Liang; Liu, Shuanglong; Liu, Yarong; Lee, Chi-Lin; Conti, Peter S; Wong, Michael K; Li, Zibo; Wang, Pin

    2013-04-01

    Liposomes constitute one of the most popular nanocarriers for the delivery of cancer therapeutics. However, since their potency is limited by incomplete drug release and inherent instability in the presence of serum components, their poor delivery occurs in certain circumstances. In this study, we address these shortcomings and demonstrate an alternative liposomal formulation, termed crosslinked multilamellar liposome (CML). With its properties of improved sustainable drug release kinetics and enhanced vesicle stability, CML can achieve controlled delivery of cancer therapeutics. CML stably encapsulated the anticancer drug doxorubicin (Dox) in the vesicle and exhibited a remarkably controlled rate of release compared to that of the unilamellar liposome (UL) with the same lipid composition or Doxil-like liposome (DLL). Our imaging study demonstrated that the CMLs were mainly internalized through a caveolin-dependent pathway and were further trafficked through the endosome-lysosome compartments. Furthermore, in vivo experiments showed that the CML-Dox formulation reduced systemic toxicity and significantly improved therapeutic activity in inhibiting tumor growth compared to that of UL-Dox or DLL-Dox. This drug packaging technology may therefore provide a new treatment option to better manage cancer and other diseases. PMID:23375392

  14. Measurement of multilamellar onion dimensions under shear using frequency domain pulsed gradient NMR

    NASA Astrophysics Data System (ADS)

    Lutti, Antoine; Callaghan, Paul T.

    2007-08-01

    We present a simple method by which the dimensions of shear-induced multilamellar vesicles (MLVs), also known as onions, can be measured during the shearing process itself. This approach is based on the use of a closely spaced train of magnetic field gradient pulses applied during a CPMG echo sequence. The CPMG train compensates flow effects while the frequency-dependence of apparent diffusion can reveal the onion size. We present here a simple phenomenological model for restricted diffusion in multilamellar vesicles, which may be used to interpret the resulting diffusion spectrum. We demonstrate this approach with MLVs formed from the lamellar phase of sodium dodecyl sulfate (SDS) in water and octanol.

  15. Ultrasound generation and high-frequency motion of magnetic nanoparticles in an alternating magnetic field: Toward intracellular ultrasound therapy?

    NASA Astrophysics Data System (ADS)

    Carrey, J.; Connord, V.; Respaud, M.

    2013-06-01

    We show theoretically that, in an inhomogeneous alternating magnetic field of frequency f, due to the alternating gradient, magnetic nanoparticles oscillate mechanically and generate ultrasound waves. This effect is maximized and better controlled if a static magnetic field is superimposed to an alternating gradient. It makes possible the generation of ultrasounds intracellularly and might also explain recent experiments in magnetic hyperthermia in which cells have been killed without any global temperature increase. Combined to an efficient targeting, it could permit ultrasound therapy with an unprecedented spatial resolution.

  16. Bcl-2 overexpression inhibits generation of intracellular reactive oxygen species and blocks adriamycin-induced apoptosis in bladder cancer cells.

    PubMed

    Kong, Chui-Ze; Zhang, Zhe

    2013-01-01

    Resistance to induction of apoptosis is a major obstacle for bladder cancer treatment. Bcl-2 is thought to be involved in anti-apoptotic signaling. In this study, we investigated the effect of Bcl-2 overexpression on apoptotic resistance and intracellular reactive oxygen species (ROS) generation in bladder cancer cells. A stable Bcl-2 overexpression cell line, BIU87-Bcl-2, was constructed from human bladder cancer cell line BIU87 by transfecting recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. The sensitivity of transfected cells to adriamycin (ADR) was assessed by MTT assay. Apoptosis was examined by flow cytometry and acridine orange fluorescence staining. Intracellular ROS was determined using flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were also investigated by the xanthinoxidase and visible radiation methods using SOD and CAT detection kits. The susceptibility of BIU87-Bcl-2 cells to ADR treatment was significantly decreased as compared with control BIU87 cells. Enhanced expression of Bcl-2 inhibited intracellular ROS generation following ADR treatment. Moreover, the suppression of SOD and CAT activity induced by ADR treatment was blocked in the BIU87-Bcl-2 case but not in their parental cells. The overexpression of Bcl-2 renders human bladder cancer cells resistant to ADR-induced apoptosis and ROS might act as an important secondary messenger in this process. PMID:23621258

  17. Soft x-ray imaging of intracellular granules of filamentous cyanobacterium generating musty smell in Lake Biwa

    NASA Astrophysics Data System (ADS)

    Takemoto, K.; Mizuta, G.; Yamamoto, A.; Yoshimura, M.; Ichise, S.; Namba, H.; Kihara, H.

    2013-10-01

    A planktonic blue-green algae, which are currently identified as Phormidium tenue, was observed by a soft x-ray microscopy (XM) for comparing a musty smell generating green strain (PTG) and a non-smell brown strain (PTB). By XM, cells were clearly imaged, and several intracellular granules which could not be observed under a light microscope were visualized. The diameter of granules was about 0.5-1 μm, and one or a few granules were seen in a cell. XM analyses showed that width of cells and sizes of intracellular granules were quite different between PTG and PTB strains. To study the granules observed by XM, transmission in more detail, transmission electron microscopy (TEM) and indirect fluorescent-antibody technique (IFA) were applied. By TEM, carboxysomes, thylakoids and polyphosphate granules were observed. IFA showed the presence of carboxysomes. Results lead to the conclusion that intracellular granules observed under XM are carboxysomes or polyphosphate granules. These results demonstrate that soft XM is effective for analyzing fine structures of small organisms such as cyanobacterium, and for discriminating the strains which generates musty smells from others.

  18. Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP

    PubMed Central

    Mani, Indra; Garg, Renu; Tripathi, Satyabha; Pandey, Kailash N.

    2015-01-01

    Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP–NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP–NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP. PMID:26374856

  19. Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool.

    PubMed

    Sannerud, Ragna; Esselens, Cary; Ejsmont, Paulina; Mattera, Rafael; Rochin, Leila; Tharkeshwar, Arun Kumar; De Baets, Greet; De Wever, Veerle; Habets, Roger; Baert, Veerle; Vermeire, Wendy; Michiels, Christine; Groot, Arjan J; Wouters, Rosanne; Dillen, Katleen; Vints, Katlijn; Baatsen, Pieter; Munck, Sebastian; Derua, Rita; Waelkens, Etienne; Basi, Guriqbal S; Mercken, Mark; Vooijs, Marc; Bollen, Mathieu; Schymkowitz, Joost; Rousseau, Frederic; Bonifacino, Juan S; Van Niel, Guillaume; De Strooper, Bart; Annaert, Wim

    2016-06-30

    γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis. PMID:27293189

  20. An Intracellular Arrangement of Histoplasma capsulatum Yeast-Aggregates Generates Nuclear Damage to the Cultured Murine Alveolar Macrophages.

    PubMed

    Pitangui, Nayla de Souza; Sardi, Janaina de Cássia Orlandi; Voltan, Aline R; Dos Santos, Claudia T; da Silva, Julhiany de Fátima; da Silva, Rosangela A M; Souza, Felipe O; Soares, Christiane P; Rodríguez-Arellanes, Gabriela; Taylor, Maria Lucia; Mendes-Giannini, Maria J S; Fusco-Almeida, Ana M

    2015-01-01

    Histoplasma capsulatum is responsible for a human systemic mycosis that primarily affects lung tissue. Macrophages are the major effector cells in humans that respond to the fungus, and the development of respiratory disease depends on the ability of Histoplasma yeast cells to survive and replicate within alveolar macrophages. Therefore, the interaction between macrophages and H. capsulatum is a decisive step in the yeast dissemination into host tissues. Although the role played by components of cell-mediated immunity in the host's defense system and the mechanisms used by the pathogen to evade the host immune response are well understood, knowledge regarding the effects induced by H. capsulatum in host cells at the nuclear level is limited. According to the present findings, H. capsulatum yeast cells display a unique architectural arrangement during the intracellular infection of cultured murine alveolar macrophages, characterized as a formation of aggregates that seem to surround the host cell nucleus, resembling a "crown." This extranuclear organization of yeast-aggregates generates damage on the nucleus of the host cell, producing DNA fragmentation and inducing apoptosis, even though the yeast cells are not located inside the nucleus and do not trigger changes in nuclear proteins. The current study highlights a singular intracellular arrangement of H. capsulatum yeast near to the nucleus of infected murine alveolar macrophages that may contribute to the yeast's persistence under intracellular conditions, since this fungal pathogen may display different strategies to prevent elimination by the host's phagocytic mechanisms. PMID:26793172

  1. Subcellular trafficking of guanylyl cyclase/natriuretic peptide receptor-A with concurrent generation of intracellular cGMP.

    PubMed

    Mani, Indra; Garg, Renu; Tripathi, Satyabha; Pandey, Kailash N

    2015-01-01

    Atrial natriuretic peptide (ANP) activates guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which lowers blood pressure and blood volume. The objective of the present study was to visualize internalization and trafficking of enhanced GFP (eGFP)-tagged NPRA (eGFP-NPRA) in human embryonic kidney-293 (HEK-293) cells, using immunofluorescence (IF) and co-immunoprecipitation (co-IP) of eGFP-NPRA. Treatment of cells with ANP initiated rapid internalization and co-localization of the receptor with early endosome antigen-1 (EEA-1), which was highest at 5 min and gradually decreased within 30 min. Similarly, co-localization of the receptor was observed with lysosome-associated membrane protein-1 (LAMP-1); however, after treatment with lysosomotropic agents, intracellular accumulation of the receptor gradually increased within 30 min. Co-IP assays confirmed that the localization of internalized receptors occurred with subcellular organelles during the endocytosis of NPRA. Rab 11, which was used as a recycling endosome (Re) marker, indicated that ∼20% of receptors recycled back to the plasma membrane. ANP-treated cells showed a marked increase in the IF of cGMP, whereas receptor was still trafficking into the intracellular compartments. Thus, after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface into endosomes, Res and lysosomes, with concurrent generation of intracellular cGMP. PMID:26374856

  2. An Intracellular Arrangement of Histoplasma capsulatum Yeast-Aggregates Generates Nuclear Damage to the Cultured Murine Alveolar Macrophages

    PubMed Central

    Pitangui, Nayla de Souza; Sardi, Janaina de Cássia Orlandi; Voltan, Aline R.; dos Santos, Claudia T.; da Silva, Julhiany de Fátima; da Silva, Rosangela A. M.; Souza, Felipe O.; Soares, Christiane P.; Rodríguez-Arellanes, Gabriela; Taylor, Maria Lucia; Mendes-Giannini, Maria J. S.; Fusco-Almeida, Ana M.

    2016-01-01

    Histoplasma capsulatum is responsible for a human systemic mycosis that primarily affects lung tissue. Macrophages are the major effector cells in humans that respond to the fungus, and the development of respiratory disease depends on the ability of Histoplasma yeast cells to survive and replicate within alveolar macrophages. Therefore, the interaction between macrophages and H. capsulatum is a decisive step in the yeast dissemination into host tissues. Although the role played by components of cell-mediated immunity in the host's defense system and the mechanisms used by the pathogen to evade the host immune response are well understood, knowledge regarding the effects induced by H. capsulatum in host cells at the nuclear level is limited. According to the present findings, H. capsulatum yeast cells display a unique architectural arrangement during the intracellular infection of cultured murine alveolar macrophages, characterized as a formation of aggregates that seem to surround the host cell nucleus, resembling a “crown.” This extranuclear organization of yeast-aggregates generates damage on the nucleus of the host cell, producing DNA fragmentation and inducing apoptosis, even though the yeast cells are not located inside the nucleus and do not trigger changes in nuclear proteins. The current study highlights a singular intracellular arrangement of H. capsulatum yeast near to the nucleus of infected murine alveolar macrophages that may contribute to the yeast's persistence under intracellular conditions, since this fungal pathogen may display different strategies to prevent elimination by the host's phagocytic mechanisms. PMID:26793172

  3. A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content.

    PubMed

    Park, Woo Hyun; Han, Yong Whan; Kim, Suhn Hee; Kim, Sung Zoo

    2007-06-01

    We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content. PMID:17382355

  4. Evidence for extracellular, but not intracellular, generation of angiotensin II in the rat adrenal zona glomerulosa

    SciTech Connect

    Urata, H.; Khosla, M.C.; Bumpus, M.; Husain, A. )

    1988-11-01

    Based on the observation that high levels of renin and angiotensin II (Ang II) are found in the adrenal zona glomerulosa (ZG), it has been postulated that Ang II is formed intracellularly by the renin-converting enzyme cascade in this tissue. To test this hypothesis, the authors examined renin-angiotensin system components in subcellular fractions of the rat adrenal ZG. Renin activity and immunoreactive-Ang II (IR-Ang II) were observed in vesicular fractions but were not colocalized. In addition, angiotensinogen, angiotensin I, and converting enzyme were not observed in the renin or IR-Ang II-containing vesicular fractions. These data do not support the hypothesis that Ang II is formed intracellularly within the renin-containing vesicles of the ZG. Rather, since modulatable renin release from adrenal ZG slices was observed and renin activity was found in dense vesicular fractions (33-39% sucrose), it is likely that Ang II formation in the ZG is extracellular and initiated by the release of vesicular renin. In ZG lysomal fractions {sup 125}I-labeled Ang II was degraded to {sup 125}I-labeled des-(Phe{sup 8})Ang II. Since Ang II antibodies do not recognize des-(Phe{sup 8})Ang II, these finding explain why IR-Ang II in the ZG is due predominantly to Ang II and not to its C-terminal immunoreactive fragments.

  5. Internalization of Proteinase 3 Is Concomitant with Endothelial Cell Apoptosis and Internalization of Myeloperoxidase with Generation of Intracellular Oxidants

    PubMed Central

    Yang, Jia Jin; Preston, Gloria A.; Pendergraft, William F.; Segelmark, Mårten; Heeringa, Peter; Hogan, Susan L.; Jennette, J. Charles; Falk, Ronald J.

    2001-01-01

    The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation. PMID:11159195

  6. Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO2-x NPs

    NASA Astrophysics Data System (ADS)

    Qiu, Yuan; Rojas, Elena; Murray, Richard A.; Irigoyen, Joseba; Gregurec, Danijela; Castro-Hartmann, Pablo; Fledderman, Jana; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio E.

    2015-04-01

    Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO2-x NPs. The brush coating does not prevent CeO2-x NPs from displaying antioxidant properties.Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell

  7. A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking1

    PubMed Central

    Hagiwara, Yukari; Kawamura, Yuki I.; Kataoka, Kosuke; Rahima, Bibi; Jackson, Raymond J.; Komase, Katsuhiro; Dohi, Taeko; Boyaka, Prosper N.; Takeda, Yoshifumi; Kiyono, Hiroshi; McGhee, Jerry R.; Fujihashi, Kohtaro

    2015-01-01

    Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity. PMID:16920941

  8. Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO(2-x) NPs.

    PubMed

    Qiu, Yuan; Rojas, Elena; Murray, Richard A; Irigoyen, Joseba; Gregurec, Danijela; Castro-Hartmann, Pablo; Fledderman, Jana; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio E

    2015-04-21

    Cerium Oxide nanoparticles (CeO(2-x) NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO(2-x) NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO(2-x) NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO(2-x) NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO(2-x) NPs. The brush coating does not prevent CeO(2-x) NPs from displaying antioxidant properties. PMID:25789459

  9. Kinetics of structural reorganizations in multilamellar photosynthetic membranes monitored by small-angle neutron scattering.

    PubMed

    Nagy, Gergely; Kovács, László; Ünnep, Renáta; Zsiros, Ottó; Almásy, László; Rosta, László; Timmins, Peter; Peters, Judith; Posselt, Dorthe; Garab, Győző

    2013-07-01

    We demonstrate the power of time-resolved small-angle neutron scattering experiments for the investigation of the structure and structural reorganizations of multilamellar photosynthetic membranes. In addition to briefly summarizing our results on thylakoid membranes isolated from higher plants and in unicellular organisms, we discuss the advantages and technical and methodological limitations of time-resolved SANS. We present a detailed and more systematical investigation of the kinetics of light-induced structural reorganizations in isolated spinach thylakoid membranes, which show how changes in the repeat distance and in the long-range order of the multilamellar membranes can be followed with a time resolution of seconds. We also present data from comparative measurements performed on thylakoid membranes isolated from tobacco. PMID:23839900

  10. Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.

    PubMed Central

    González-Flecha, B; Demple, B

    1994-01-01

    Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a

  11. Flow Properties of Multilamellar Droplets in AOT/Brine/Glycerol Mixtures

    NASA Astrophysics Data System (ADS)

    Ishii, Akiko; Pons, Ramon; Ushiki, Hideharu; Rouch, Jacques; Letamendia, Louis

    2004-09-01

    The properties of lamellar solutions formed in a quaternary mixture made of brine, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and glycerol have been studied under shear flow. As in AOT, brine pseudo-ternary system, the lamellar solutions exhibit a structural transition above a threshold shear rate, and form quasi-monodispersed multilamellar droplets. In this regime, the solutions are shear thinning. Both the viscosity and the droplet size scale with the shear rate with exponents respectively equal to -0.7 and -0.4. This last value is smaller than the one, 0.5, reported in the ternary systems. Tentative explanations of our experimental results are given.

  12. Permeability of iodide in multilamellar liposomes modeled by two compartments and a reservoir.

    PubMed

    Schullery, S E

    1977-07-14

    A previously published rate law for the diffusion of iodide from multilamellar egg phosphatidylcholine liposomes (Schullery, S.E. (1975) Chem. Phys. Lipids 14, 49-58) is fitted to the relatively simple mathematical model of two compartments in series with a reservoir. All of the inner liposome compartments are assumed to behave as effectively one compartment in series with the liposome's outermost compartment. Based on this model, reasonable values are calculated for the fraction of the total solution trapped by liposomes which is in the outermost liposome compartment, 17%, and the permeability coefficient of iodide against isotonic, mixed iodide-chloride solution, 2-10(-9) cm/s. PMID:884087

  13. IL-12 Delivered Intratumorally by Multilamellar Liposomes Reactivates Memory T Cells in Human Tumor Microenvironments

    PubMed Central

    Simpson-Abelson, Michelle R.; Purohit, Vivek S.; Pang, Wing Man; Iyer, Vandana; Odunsi, Kunle; Demmy, Todd L; Yokota, Sandra J.; Loyall, Jenni L.; Kelleher, Raymond J.; Balu-Iyer, Sathy; Bankert, Richard B.

    2009-01-01

    Using a novel loading technique, IL-12 is reported here to be efficiently encapsulated within large multilamellar liposomes. The preclinical efficacy of the cytokine loaded liposomes to deliver IL-12 into human tumors and to reactive tumor-associated T cells in situ is tested using a human tumor xenograft model. IL-12 is released in vivo from these liposomes in a biologically active form when injected into tumor xenografts that are established by the subcutaneous implantation of non-disrupted pieces of human lung, breast or ovarian tumors into immunodeficient mice. The histological architecture of the original tumor tissue, including tumor-associated leukocytes, tumor cells and stromal cells is preserved anatomically and the cells remain functionally responsive to cytokines in these xenografts. The local and sustained release of IL-12 into the tumor microenvironment reactivates tumor-associated quiescent effector memory T cells to proliferate, produce and release IFN-γ resulting in the killing of tumor cells in situ. Very little IL-12 is detected in the serum of mice for up to 5 days after an intratumoral injection of the IL-12 liposomes. We conclude that IL-12 loaded large multilamellar liposomes provide a safe method for the local and sustained delivery of IL-12 to tumors and a therapeutically effective way of reactivating existing tumor-associated T cells in human solid tumor microenvironments. The potential of this local in situ T cell re-stimulation to induce a systemic anti-tumor immunity is discussed. PMID:19395317

  14. Dehydration of multilamellar fatty acid membranes: Towards a computational model of the stratum corneum

    NASA Astrophysics Data System (ADS)

    MacDermaid, Christopher M.; DeVane, Russell H.; Klein, Michael L.; Fiorin, Giacomo

    2014-12-01

    The level of hydration controls the cohesion between apposed lamellae of saturated free fatty acids found in the lipid matrix of stratum corneum, the outermost layer of mammalian skin. This multilamellar lipid matrix is highly impermeable to water and ions, so that the local hydration shell of its fatty acids may not always be in equilibrium with the acidity and relative humidity, which significantly change over a course of days during skin growth. The homeostasis of the stratum corneum at each moment of its growth likely requires a balance between two factors, which affect in opposite ways the diffusion of hydrophilic species through the stratum corneum: (i) an increase in water order as the lipid lamellae come in closer contact, and (ii) a decrease in water order as the fraction of charged fatty acids is lowered by pH. Herein molecular dynamics simulations are employed to estimate the impact of both effects on water molecules confined between lamellae of fatty acids. Under conditions where membrane undulations are energetically favorable, the charged fatty acids are able to sequester cations around points of contact between lamellae that are fully dehydrated, while essentially maintaining a multilamellar structure for the entire system. This observation suggests that the undulations of the fatty acid lamellae control the diffusion of hydrophilic species through the water phase by altering the positional and rotational order of water molecules in the embedded/occluded "droplets."

  15. Multilamellar vesicle formation from a planar lamellar phase under shear flow.

    PubMed

    Gentile, Luigi; Behrens, Manja A; Porcar, Lionel; Butler, Paul; Wagner, Norman J; Olsson, Ulf

    2014-07-22

    The formation of multilamellar vesicles (MLVs) from the lamellar phase of nonionic surfactant system C12E5/D2O under shear flow is studied by time-resolved small angle neutron and light scattering during shear flow. A novel small angle neutron scattering sample environment enables the tracking of the lamellae alignment in the velocity-velocity gradient (1-2) plane during MLV formation, which was tracked independently using flow small angle light scattering commensurate with rheology. During the lamellar-to-multilamellar vesicle transition, the primary Bragg peak from the lamellar ordering was observed to tilt, and this gradually increased with time, leading to an anisotropic pattern with a primary axis oriented at ∼25° relative to the flow direction. This distorted pattern persists under flow after MLV formation. A critical strain and critical capillary number based on the MLV viscosity are demonstrated for MLV formation, which is shown to be robust for other systems as well. These novel measurements provide fundamentally new information about the flow orientation of lamellae in the plane of flow that cannot be anticipated from the large body of previous literature showing nearly isotropic orientation in the 2,3 and 1,3 planes of flow. These observations are consistent with models for buckling-induced MLV formation but suggest that the instability is three-dimensional, thereby identifying the mechanism of MLV formation in simple shear flow. PMID:24983325

  16. Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

    NASA Astrophysics Data System (ADS)

    Moon, James J.; Suh, Heikyung; Bershteyn, Anna; Stephan, Matthias T.; Liu, Haipeng; Huang, Bonnie; Sohail, Mashaal; Luo, Samantha; Ho Um, Soong; Khant, Htet; Goodwin, Jessica T.; Ramos, Jenelyn; Chiu, Wah; Irvine, Darrell J.

    2011-03-01

    Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.

  17. Global SAXS Data Analysis for Multilamellar Vesicles: Evolution of the Scattering Density Profile (SDP) Model

    SciTech Connect

    Heftberger, Peter; Kollmitzer, Benjamin; Heberle, Frederick A; Pan, Jianjun; Rappolt, Michael; Amenitsch, Heinz; Kucerka, Norbert; Katsaras, John; Pabst, georg

    2014-01-01

    The highly successful scattering density profile (SDP) model, used to jointly analyze small-angle X-ray and neutron scattering data from unilamellar vesicles, has been adapted for use with data from fully hydrated, liquid crystalline multilamellar vesicles (MLVs). Using a genetic algorithm, this new method is capable of providing high-resolution structural information, as well as determining bilayer elastic bending fluctuations from standalone X-ray data. Structural parameters such as bilayer thickness and area per lipid were determined for a series of saturated and unsaturated lipids, as well as binary mixtures with cholesterol. The results are in good agreement with previously reported SDP data, which used both neutron and X-ray data. The inclusion of deuterated and non-deuterated MLV neutron data in the analysis improved the lipid backbone information but did not improve, within experimental error, the structural data regarding bilayer thickness and area per lipid.

  18. Influence of cholesterol and ceramide VI on the structure of multilamellar lipid membranes at water exchange

    SciTech Connect

    Ryabova, N. Yu. Kiselev, M. A.; Balagurov, A. M.

    2010-05-15

    The structural changes in the multilamellar lipid membranes of dipalmitoylphosphatidylcholine (DPPC)/cholesterol and DPPC/ceramide VI binary systems during hydration and dehydration have been studied by neutron diffraction. The effect of cholesterol and ceramide on the kinetics of water exchange in DPPC membranes is characterized. Compared to pure DPPC, membranes of binary systems swell faster during hydration (with a characteristic time of {approx}30 min). Both compounds, ceramide VI and cholesterol, similarly affect the hydration of DPPC membranes, increasing the repeat distance due to the bilayer growth. However, in contrast to cholesterol, ceramide significantly reduces the thickness of the membrane water layer. The introduction of cholesterol into a DPPC membrane slows down the change in the parameters of the bilayer internal structure during dehydration. In the DPPC/ceramide VI/cholesterol ternary system (with a molar cholesterol concentration of 40%), cholesterol is partially released from the lamellar membrane structure into the crystalline phase.

  19. Codelivery of Chemotherapeutics via Crosslinked Multilamellar Liposomal Vesicles to Overcome Multidrug Resistance in Tumor

    PubMed Central

    Joo, Kye-Il; Wong, Michael K.; Wang, Pin

    2014-01-01

    Multidrug resistance (MDR) is a significant challenge to effective cancer chemotherapy treatment. However, the development of a drug delivery system that allows for the sustained release of combined drugs with improved vesicle stability could overcome MDR in cancer cells. To achieve this, we have demonstrated codelivery of doxorubicin (Dox) and paclitaxel (PTX) via a crosslinked multilamellar vesicle (cMLV). This combinatorial delivery system achieves enhanced drug accumulation and retention, in turn resulting in improved cytotoxicity against tumor cells, including drug-resistant cells. Moreover, this delivery approach significantly overcomes MDR by reducing the expression of P-glycoprotein (P-gp) in cancer cells, thus improving antitumor activity in vivo. Thus, by enhancing drug delivery to tumors and lowering the apoptotic threshold of individual drugs, this combinatorial delivery system represents a potentially promising multimodal therapeutic strategy to overcome MDR in cancer therapy. PMID:25330237

  20. Mechanism of electroinduced ionic species transport through a multilamellar lipid system.

    PubMed Central

    Chizmadzhev, Y A; Zarnitsin, V G; Weaver, J C; Potts, R O

    1995-01-01

    A theoretical model for electroporation of multilamellar lipid system due to a series of large electrical pulses is presented and then used to predict the functional dependence of the transport of charged molecules. Previously, electroporation has been considered only for single bilayer systems such as artificial planar bilayer membranes and cell membranes. The former have been extensively studied with respect to electrical and mechanical behavior, and the latter with respect to molecular transport. Recent experimental results for both molecular transport and electrical resistance changes in the stratum corneum (SC) suggest that electroporation also occurs in the multilamellar lipid membranes of the SC. In addition, there is the possibility that other skin structures (the "appendages") also experience electroporation. A compartment model is introduced to describe the transport of charged species across the SC, and the predicted dependence is compared with available data. In this model, the SC is assumed to contain many hydrophilic compartments in series separated by boundary bilayers, so that these compartments become connected only upon electroporation. Two limiting cases for the transport of charged molecules are considered: (1) transport along tortuous inter-bilayer pathways in each compartment, followed by transport across individual boundary bilayers due to electroporation, and (2) transport along straight-through pathways in the boundary bilayers with fast mixing in each compartment, which includes the interior space of corneocytes. Both models were fitted to the experimental data. The large electropore radius (rt approximately 200 A) and porated fractional area (ft approximately 10(-3) obtained from the fitting for the tortuous model relative to the more reasonable values obtained for the straight-through model (rs approximately 4 A, fs approximately 10(-6) suggest that the latter is a more realistic description of electroinduced transport of ionized species

  1. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  2. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

    PubMed

    Uchiyama, Hidefumi; Zhao, Qing-Li; Hassan, Mariame Ali; Andocs, Gabor; Nojima, Nobuyuki; Takeda, Keigo; Ishikawa, Kenji; Hori, Masaru; Kondo, Takashi

    2015-01-01

    Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an

  3. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu

    PubMed Central

    Uchiyama, Hidefumi; Zhao, Qing-Li; Hassan, Mariame Ali; Andocs, Gabor; Nojima, Nobuyuki; Takeda, Keigo; Ishikawa, Kenji; Hori, Masaru; Kondo, Takashi

    2015-01-01

    Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an

  4. The interaction of atmospheric pressure plasma jets with cancer and normal cells: generation of intracellular reactive oxygen species and changes of the cell proliferation and cell cycle

    NASA Astrophysics Data System (ADS)

    Chung, Tae Hun; Joh, Hea Min; Kim, Sun Ja; Leem, Sun Hee

    2013-09-01

    The possibility of atmospheric pressure plasmas is emerging as a candidate in cancer therapy. The primary role is played by reactive oxygen species (ROS), UV photons, charged particles and electric fields. Among them, intracellular ROS induced by plasma are considered to be the key constituents that induce cellular changes and apoptosis. In this study, the effects of atmospheric pressure plasma jet on cancer cells (human lung carcinoma cells) and normal cells (embryonic kidney cells and bronchial epithelial cells) were investigated. The plasma treatment was performed under different working gases, applied voltages, gas flow rates, and with and without additive oxygen flow. Using a detection dye, we observed that plasma exposure leads to the increase of the intracellular ROS and that the intracellular ROS production can be controlled by plasma parameters. A significant ROS generation was induced by plasma exposure on cancer cells and the overproduction of ROS contributes to the reduced cell proliferation. Normal cells were observed to be less affected by the plasma-mediated ROS and cell proliferation was less changed. The plasma treatment also resulted in the alteration of the cell cycle that contributes to the induction of apoptosis in cancer cells. The selective effect on cancer and normal cells provides a promising prospect of cold plasma as cancer therapy. This work was supported by the National Research Foundation of Korea under Contract No. 2012R1A1A2002591 and 2012R1A1A3010213.

  5. Enhancement of Lateral Diffusion in Catanionic Vesicles during Multilamellar-to-Unilamellar Transition.

    PubMed

    Mitra, S; Sharma, V K; Garcia-Sakai, V; Orecchini, A; Seydel, T; Johnson, M; Mukhopadhyay, R

    2016-04-21

    Catanionic vesicles are formed spontaneously by mixing cationic and anionic dispersions in aqueous solution in suitable conditions. Because of spontaneity in formation, long-term stability, and easy modulation of size and charge, they have numerous advantages over conventional lipid-based vesicles. The dynamics of such vesicles is of interest in the field of biomedicine, as they can be used to deliver drug molecules into the cell membrane. Dynamics of catanionic vesicles based on sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) have been studied using incoherent elastic and quasielastic neutron scattering (QENS) techniques. Neutron scattering experiments have been carried out on two backscattering spectrometers, IRIS and IN16B, which have different energy resolutions and energy transfer windows. An elastic fixed-window scan carried out using IN16B shows a phase transition at ∼307 K during the heating cycle, whereas on cooling the transition occurred at ∼294 K. DSC results are found to be in close agreement with the elastic scan data. This transition is ascribed to a structural rearrangement from a multilamellar to a unilamellar phase [ Andreozzi J. Phys. Chem. B 2010 , 114 , 8056 - 8060 ]. It is found that a model in which the surfactant molecules undergo both lateral and internal motions can describe the QENS data quite well. While the data from IRIS have contributions from both dynamical processes, the data from IN16B probe only lateral motions, as the internal motions are too fast for the energy window of the spectrometer. It is found that, through the transition, the fraction of surfactant molecules undergoing lateral motion increases of a factor of 2 from the multilamellar to the unilamellar phase, indicating an enhanced fluidity of the latter. The lateral motion is found to be Fickian in nature, while the internal motion has been described by a localized translational diffusion model. The results reported here could have direct

  6. Multi-lamellar vesicle formation in a long-chain nonionic surfactant: C16E4/D2O system.

    PubMed

    Gentile, Luigi; Mortensen, Kell; Rossi, Cesare Oliviero; Olsson, Ulf; Ranieri, Giuseppe A

    2011-10-01

    The temperature dependent rheological and structural behavior of a long-chain C(16)E(4) (tetraethylene glycol monohexadecyl ether) surfactant in D(2)O has been studied within the regime of low shear range. In the absence of shear flow, the system forms a lamellar liquid crystalline phase at relatively high temperatures. The present paper reports on the shear-induced multi-lamellar vesicle (MLV) formation in C(16)E(4)/D(2)O at 40 wt.% of surfactant in the temperature range of 40-55 °C. The transition from planar lamellar structure to multi-lamellar vesicles has been investigated by time-resolved experiments combining rheology and nuclear magnetic resonance (rheo-NMR), rheo small-angle neutron scattering (rheo-SANS) and rheometry. The typical transient viscosity behavior of MLV formation has been discovered at low shear rate value of 0.5s(-1). PMID:21767850

  7. Encapsulation of rutin and naringenin in multilamellar vesicles for optimum antioxidant activity.

    PubMed

    Kerdudo, Audrey; Dingas, Alexandre; Fernandez, Xavier; Faure, Chrystel

    2014-09-15

    Rutin and naringenin, two phenolic compounds with antioxidant properties were encapsulated in lipid-based onion-type multilamellar vesicles (MLVs). After vesicles formation, the free, adsorbed/encapsulated analytes were well separated with size exclusion chromatography (SEC), and rutin and naringenin were quantified with UV-HPLC at 258 nm and 290 nm. A mathematical model was developed to separately calculate the encapsulation and the adsorption yields of both phenols. Naringenin was shown to be poorly encapsulated (<10%) but highly adsorbed on MLVs surface (>60%) whatever MLVs composition. Conversely, rutin showed high encapsulation efficiency (>60%). Entrapment of rutin was proved to be efficient since no leak was observed within 30 days in concentrated MLVs phase, while 16.0±0.3% of rutin was still encapsulated after 30 days when MLVs were diluted in water. Free rutin broke up into quercetin while the encapsulated one remained stable. DPPH assay confirmed that only free and adsorbed rutin participated in antioxidant activity. PMID:24767021

  8. Interactions of tamoxifen with distearoyl phosphatidylcholine multilamellar vesicles: FTIR and DSC studies

    NASA Astrophysics Data System (ADS)

    Bilge, Duygu; Sahin, Ipek; Kazanci, Nadide; Severcan, Feride

    2014-09-01

    Interactions of a non-steroidal antiestrogen drug, tamoxifen (TAM), with distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) multilamellar liposomes (MLVs) were investigated as a function of drug concentration (1-15 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). FTIR spectroscopy results show that increasing TAM concentrations (except 1 mol%) increased the wavenumbers of the CH2 stretching modes, implying an disordering effect for DSPC MLVs both in the gel and liquid crystalline phases. The bandwidth values of the CH2 stretchings except for 1 mol% increased when TAM concentrations increased for DSPC liposomes, indicating an increase in the dynamics of liposomes. The Cdbnd O stretching and PO2- antisymmetric double bond stretching bands were analyzed to study interactions of TAM with head groups of lipids. As the concentrations of TAM increased, dehydration occurred around these functional groups in the polar part of the lipids. The DSC studies on thermal properties of DSPC lipids indicate that TAM eliminated the pre transition, shifted the main phase transition to lower temperatures and broadened the phase transition curve of the liposomes.

  9. Preventive Effects of Multi-Lamellar Emulsion on Low Potency Topical Steroid Induced Local Adverse Effect

    PubMed Central

    Sul, Geun Dong; Park, Hyun Jung; Bae, Jong Hwan; Hong, Keum Duck; Park, Byeong Deog; Chun, Jaesun; Jeong, Se Kyoo; Lee, Seung Hun; Ahn, Sung Ku

    2013-01-01

    Background Topical steroid treatment induces diverse local Wand systemic adverse effects. Several approaches have been tried to reduce the steroid-induced adverse effects. Simultaneous application of physiological lipid mixture is also suggested. Objective Novel vehicles for topical glucocorticoids formulation were evaluated for the efficacy of reducing side-effects and the drug delivery properties of desonide, a low potency topical steroid. Methods Transcutaneous permeation and skin residual amount of desonide were measured using Franz diffusion cells. The in vivo anti-inflammatory activity was evaluated using murine model. Results Topical steroids formulation containing desonide, in either cream or lotion form, were prepared using multi-lamellar emulsion (MLE), and conventional desonide formulations were employed for comparison. MLE formulations did not affect the anti-inflammatory activity of the desonide in phobol ester-induced skin inflammation model, compared with conventional formulations. While the penetrated amounts of desonide were similar for all the tested formulations at 24 hours after application, the increased lag time was observed for the MLE formulations. Interestingly, residual amount of desonide in epidermis was significantly higher in lotion type MLE formulation. Steroid-induced adverse effects, including permeability barrier function impairment, were partially prevented by MLE formulation. Conclusion Topical desonide formulation using MLE as a vehicle showed a better drug delivery with increased epidermal retention. MLE also partially prevented the steroid-induced side effects, such as skin barrier impairment. PMID:23467730

  10. Stable and fluid multilayer phospholipid-silica thin films: mimicking active multi-lamellar biological assemblies.

    PubMed

    Gupta, Gautam; Iyer, Srinivas; Leasure, Kara; Virdone, Nicole; Dattelbaum, Andrew M; Atanassov, Plamen B; López, Gabriel P

    2013-06-25

    Phospholipid-based nanomaterials are of interest in several applications including drug delivery, sensing, energy harvesting, and as model systems in basic research. However, a general challenge in creating functional hybrid biomaterials from phospholipid assemblies is their fragility, instability in air, insolubility in water, and the difficulty of integrating them into useful composites that retain or enhance the properties of interest, therefore limiting there use in integrated devices. We document the synthesis and characterization of highly ordered and stable phospholipid-silica thin films that resemble multilamellar architectures present in nature such as the myelin sheath. We have used a near room temperature chemical vapor deposition method to synthesize these robust functional materials. Highly ordered lipid films are exposed to vapors of silica precursor resulting in the formation of nanostructured hybrid assemblies. This process is simple, scalable, and offers advantages such as exclusion of ethanol and no (or minimal) need for exposure to mineral acids, which are generally required in conventional sol-gel synthesis strategies. The structure of the phospholipid-silica assemblies can be tuned to either lamellar or hexagonal organization depending on the synthesis conditions. The phospholipid-silica films exhibit long-term structural stability in air as well as when placed in aqueous solutions and maintain their fluidity under aqueous or humid conditions. This platform provides a model for robust implementation of phospholipid multilayers and a means toward future applications of functional phospholipid supramolecular assemblies in device integration. PMID:23706112

  11. Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

    PubMed

    Trigui, Hana; Paquet, Valérie E; Charette, Steve J; Faucher, Sébastien P

    2016-05-01

    Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence ofC. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized thatC. jejunicould be packaged under aerobic conditions by the amoebaAcanthamoeba castellaniior the ciliateTetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellaniidid not produce MLBs containingC. jejuni In contrast, when incubated withT. pyriformis,C. jejuniwas ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics ofC. jejuniculturability showed that packaging increased the survival ofC. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests thatT. pyriformismay increase the risk of persistence ofC. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging. PMID:26921427

  12. Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

    PubMed

    Denoncourt, Alix M; Paquet, Valérie E; Sedighi, Ahmadreza; Charette, Steve J

    2016-01-01

    Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs. PMID:27340834

  13. Codelivery of Doxorubicin and Paclitaxel by Cross-Linked Multilamellar Liposome Enables Synergistic Antitumor Activity

    PubMed Central

    2015-01-01

    Combining chemotherapeutics is a promising method of improving cancer treatment; however, the clinical success of combination therapy is limited by the distinct pharmacokinetics of combined drugs, which leads to nonuniform distribution. In this study, we report a new robust approach to load two drugs with different hydrophilicities into a single cross-linked multilamellar liposomal vesicle (cMLV) to precisely control the drug ratio that reaches the tumor in vivo. The stability of cMLVs improves the loading efficiency and sustained release of doxorubicin (Dox) and paclitaxel (PTX), maximizing the combined therapeutic effect and minimizing the systemic toxicity. Furthermore, we show that the cMLV formulation maintains specific drug ratios in vivo for over 24 h, enabling the ratio-dependent combination synergy seen in vitro to translate to in vivo antitumor activity and giving us control over another parameter important to combination therapy. This combinatorial delivery system may provide a new strategy for synergistic delivery of multiple chemotherapeutics with a ratiometric control over encapsulated drugs to treat cancer and other diseases. PMID:24673622

  14. Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum

    PubMed Central

    Denoncourt, Alix M.; Paquet, Valérie E.; Sedighi, Ahmadreza; Charette, Steve J.

    2016-01-01

    Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs. PMID:27340834

  15. rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures.

    PubMed

    Domingues, Marco M; Bianconi, M Lucia; Barbosa, Leandro R S; Santiago, Patrícia S; Tabak, Marcel; Castanho, Miguel A R B; Itri, Rosangela; Santos, Nuno C

    2013-11-01

    rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death. PMID:23792068

  16. Estimation of mechanical strength of unilamellar and multilamellar AOT/water vesicles and their rupture using micropipet aspiration.

    PubMed

    Sagar, G Hema; Bellare, Jayesh R

    2009-10-22

    Vesicles prepared from surfactant sodium dioctyl sulfosuccinate (AOT) were characterized by micropipet aspiration for determining membrane bending rigidity and area expansion modulus and mechanism of rupture. Unilamellar vesicles (ULV) and multilamellar vesicles (MLV) were studied. The mechanical properties calculated using micropipet aspiration for ULV were found to be 5-10 k(B)T, K(a) = 100 +/- 20 mN/m and for MLV were 8-15 k(B)T, K(a) = 120 +/- 30 mN/m. These properties fall with the range of lipid (PC) membrane measurements (values). However, the membrane rigidity of multilamellar vesicles was found to be approximately 3 times larger than that of unilamellar vesicles. The apparent area expansion moduli of multilamellar vesicles are of the order 1.4 times, sustained far greater areal strain before rupture compared to that of unilamellar vesicles. A dynamic structural change in MLV is demonstrated upon stress by micropipet aspiration. MLV at stress undergoes various stages of deformation. The fluctuation in size and shape of MLV led to separation of bilayers from the stack and decrease in vesicle diameter facilitating in formation of new equilibrium MLV, for it to sustain the specified membrane tension, a new mechanism that is demonstrated experimentally. PMID:19764699

  17. Evaluation of extra- and intracellular OH radical generation, cancer cell injury, and apoptosis induced by a non-thermal atmospheric-pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Ninomiya, Kazuaki; Ishijima, Tatsuo; Imamura, Masatoshi; Yamahara, Takayuki; Enomoto, Hiroshi; Takahashi, Kenji; Tanaka, Yasunori; Uesugi, Yoshihiko; Shimizu, Nobuaki

    2013-10-01

    In this study, we investigated the effects of a non-thermal atmospheric-pressure plasma jet inducing extracellular and intracellular OH radical generation as well as cell injury and apoptosis for the cultured human breast cancer cells. Increased OH radical generation in the extracellular culture medium (liquid phase) was observed with increased irradiation time, distance to the liquid surface, and voltage. From the voltage-response relationships for two breast cancer cell lines (invasive MDA-MB-231 cells and non-invasive MCF-7 cells) and normal breast cells (HMEC), the half-maximal effective peak-to-peak voltage (EV50) values were 16.7 ± 0.3 kV, 15.0 ± 0.4 kV and 11.2 ± 0.7 kV for MDA-MB-231, MCF-7 and HMEC cells, respectively. This indicated that there was almost no selective cancer cell injury induced by plasma jet irradiation under these conditions. Compared with control condition without a plasma jet, intracellular OH radical accumulation and apoptotic cells were observed with a plasma jet using conditions that induced injury to 50% of cells irrespective of the cancer cell line.

  18. Multimodal function of the sweet taste receptor expressed in pancreatic β-cells: generation of diverse patterns of intracellular signals by sweet agonists.

    PubMed

    Nakagawa, Yuko; Nagasawa, Masahiro; Mogami, Hideo; Lohse, Martin; Ninomiya, Yuzo; Kojima, Itaru

    2013-01-01

    The sweet taste receptor is expressed in the taste bud and is activated by numerous sweet molecules with diverse chemical structures. It is, however, not known whether these sweet agonists induce a similar cellular response in target cells. Using MIN6 cells, a pancreatic β-cell line expressing endogenous sweet taste receptor, we addressed this question by monitoring changes in cytoplasmic Ca2+ ([Ca2+]i) and cAMP ([cAMP]i) induced by four sweet taste receptor agonists. Glycyrrhizin evoked sustained elevation of [Ca2+]i but [cAMP]i was not affected. Conversely, an artificial sweetener saccharin induced sustained elevation of [cAMP]i but did not increase [Ca2+]i. In contrast, sucralose and acesulfame K induced rapid and sustained increases in both [Ca2+]i and [cAMP]i. Although the latter two sweeteners increased [Ca2+]i and [cAMP]i, their actions were not identical: [Ca2+]i response to sucralose but not acesulfame K was inhibited by gurmarin, an antagonist of the sweet taste receptor which blocks the gustducin-dependent pathway. In addition, [Ca2+]i response to acesulfame K but not to sucralose was resistant to a Gq inhibitor. These results indicate that four types of sweeteners activate the sweet taste receptor differently and generate distinct patterns of intracellular signals. The sweet taste receptor has amazing multimodal functions producing multiple patterns of intracellular signals. PMID:23933592

  19. Enhancing Radiotherapy by Lipid Nanocapsule-Mediated Delivery of Amphiphilic Gold Nanoparticles to Intracellular Membranes

    PubMed Central

    Yang, Yu-Sang; Carney, Randy P.; Stellacci, Francesco; Irvine, Darrell J.

    2014-01-01

    Amphiphilic gold nanoparticles (amph-NPs), composed of gold cores surrounded by an amphiphilic mixed organic ligand shell, are capable of embedding within and traversing lipid membranes. Here we describe a strategy using crosslink-stabilized lipid nanocapsules (NCs) as carriers to transport such membrane-penetrating particles into tumor cells and promote their transfer to intracellular membranes for enhanced radiotherapy of cancer. We synthesized and characterized interbilayer-crosslinked multilamellar lipid vesicles (ICMVs) carrying amph-NPs embedded in the capsule walls, forming Au-NCs. Confocal and electron microscopies revealed that the intracellular distribution of amph-NPs within melanoma and breast tumor cells following uptake of free particles v.s. Au-NCs was quite distinct, and that amph-NPs initially delivered into endosomes by Au-NCs transferred over a period of hours to intracellular membranes through tumor cells, with greater intracellular spread in melanoma cells than breast carcinoma cells. Clonogenic assays revealed that Au-NCs enhanced radiotherapeutic killing of melanoma cells. Thus, multilamellar lipid capsules may serve as an effective carrier to deliver amphiphilic gold nanoparticles to tumors, where the membrane-penetrating properties of these materials can significantly enhance the efficacy of frontline radiotherapy treatments. PMID:25123510

  20. Plumbagin Inhibits Proliferative and Inflammatory Responses of T Cells Independent of ROS Generation But by Modulating Intracellular Thiols

    PubMed Central

    Checker, Rahul; Sharma, Deepak; Sandur, Santosh K.; Subrahmanyam, G.; Krishnan, Sunil; Poduval, T.B.; Sainis, K.B.

    2011-01-01

    Plumbagin inhibited activation, proliferation, cytokine production, and graft-versus-host disease in lymphocytes and inhibited growth of tumor cells by suppressing nuclear factor-κB (NF-κB). Plumbagin was also shown to induce reactive oxygen species (ROS) generation in tumor cells via an unknown mechanism. Present report describes a novel role of cellular redox in modulation of immune responses in normal lymphocytes by plumbagin. Plumbagin depleted glutathione (GSH) levels that led to increase in ROS generation. The decrease in GSH levels was due to direct reaction of plumbagin with GSH as evinced by mass spectrometric and HPLC analysis. Further, addition of plumbagin to cells resulted in decrease in free thiol groups on proteins and increase in glutathionylation of proteins. The suppression of mitogen-induced T-cell proliferation and cytokine (IL-2/IL-4/IL-6/IFN-γ) production by plumbagin was abrogated by thiol antioxidants but not by non-thiol antioxidants confirming that thiols but not ROS play an important role in biological activity of plumbagin. Plumbagin also abrogated mitogen-induced phosphorylation of ERK, IKK, and degradation of IκB-α. However, it did not affect phosphorylation of P38, JNK, and AKT. Our results for the first time show that antiproliferative effects of plumbagin are mediated by modulation of cellular redox. These results provide a rationale for application of thiol-depleting agents as anti-inflammatory drugs. PMID:20564204

  1. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  2. Intracellular Generation of ROS by 3,5-Dimethylaminophenol: Persistence, Cellular Response, and Impact of Molecular Toxicity

    PubMed Central

    Chao, Ming-Wei; Erkekoglu, Pinar; Tseng, Chia-Yi; Ye, Wenjie; Trudel, Laura J.; Skipper, Paul L.; Tannenbaum, Steven R.; Wogan, Gerald N.

    2014-01-01

    Epidemiological studies have demonstrated extensive human exposure to the monocyclic aromatic amines, particularly to 3,5-dimethylaniline, and found an association between exposure to these compounds and risk for bladder cancer. Little is known about molecular mechanisms that might lead to the observed risk. We previously suggested that the hydroxylated 3,5-dimethylaniline metabolite, 3,5-dimethylaminophenol (3,5-DMAP), played a central role in effecting genetic change through the generation of reactive oxygen species (ROS) in a redox cycle with 3,5-dimethylquinoneimine. Experiments here characterize ROS generation by 3,5-DMAP exposure in nucleotide repair-proficient and -deficient Chinese hamster ovary cells as a function of time. Besides, various cellular responses discussed herein indicate that ROS production is the principal cause of cytotoxicity. Fluorescence microscopy of cells exposed to 3,5-DMAP confirmed that ROS production occurs in the nuclear compartment, as suggested by a previous study demonstrating covalent linkage between 3,5-DMAP and histones. 3,5-DMAP was also compared with 3,5-dimethylhydroquinone to determine whether substitution of one of the phenolic hydroxyl groups by an amino group had a significant effect on some of the investigated parameters. The comparatively much longer duration of observable ROS produced by 3,5-DMAP (7 vs. 1 day) provides further evidence that 3,5-DMAP becomes embedded in the cellular matrix in a form capable of continued redox cycling. 3,5-DMAP also induced dose-dependent increase of H2O2 and ·OH, which were determined as the major free radicals contributing to the cytotoxicity and apoptosis mediated via caspase-3 activation. Overall, this study provides insight into the progression of alkylaniline-induced toxicity. PMID:24973092

  3. Intracellular microlasers

    PubMed Central

    Humar, Matjaž; Yun, Seok Hyun

    2015-01-01

    Optical microresonators1 which confine light within a small cavity are widely exploited for various applications ranging from the realization of lasers2 and nonlinear devices3, 4, 5 to biochemical and optomechanical sensing6, 7, 8, 9, 10, 11. Here we employ microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explored two distinct types of microresonators: soft and hard, that support whispering-gallery modes (WGM). Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (~500 pN/μm2) and its dynamic fluctuations at a sensitivity of 20 pN/μm2 (20 Pa). In a second form, WGMs within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes. PMID:26417383

  4. Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization.

    PubMed

    Stefani, Christian; Al-Eisawi, Zaynab; Jansson, Patric J; Kalinowski, Danuta S; Richardson, Des R

    2015-11-01

    Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular (64)Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones). PMID:26335599

  5. Passive entry of CO2 and its energy-dependent intracellular conversion to HCO3- in cyanobacteria are driven by a photosystem I-generated deltamuH+.

    PubMed

    Tchernov, D; Helman, Y; Keren, N; Luz, B; Ohad, I; Reinhold, L; Ogawa, T; Kaplan, A

    2001-06-29

    CO(2) entry into Synechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO(2) uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO3(-). Dependence of CO(2) uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO(2) uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a DeltamuH(+) generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO(2) uptake. Under low light intensity, the rate of CO(2) uptake by a high-CO(2)-requiring mutant of Synechococcus sp. PCC7942, at a CO(2) concentration below its threshold for CO(2) fixation, was higher than that of the wild type. At saturating light intensity, net CO(2) uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO(2) to HCO3(-). These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO(2) to HCO3(-). PMID:11297562

  6. Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

    PubMed

    McCauliff, Leslie A; Xu, Zhi; Li, Ran; Kodukula, Sarala; Ko, Dennis C; Scott, Matthew P; Kahn, Peter C; Storch, Judith

    2015-11-01

    The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment. PMID:26296895

  7. Quantitative study of the encapsulation of glucose oxidase into multilamellar vesicles and its effect on enzyme activity

    NASA Astrophysics Data System (ADS)

    Olea, David; Faure, Chrystel

    2003-09-01

    The encapsulation of glucose oxidase (GOx) into onion-type multilamellar vesicles is studied and compared to that of GOx into liposomes. The enzyme was shown not to be affected by encapsulation as evidenced by the complete recovery of its activity after being freed. An ˜15% increase of GOx activity was conferred by confinement in onions in the 30-50 °C temperature range. Entrapment of GOx in onions was proved to be effective since a maximum of 10% leak was measured after 45 days of encapsulation. The encapsulation yield, which reaches 80%, and the number of encapsulated enzyme molecules per onion (1000 GOx molecules) were found to be much higher than for liposomes. The effect of onion composition on the encapsulation yield was determined and predicted by a thermodynamic model applied to the lipids-GOx-phosphate buffer system.

  8. Structural information from multilamellar liposomes at full hydration: Full q-range fitting with high quality x-ray data

    NASA Astrophysics Data System (ADS)

    Pabst, Georg; Rappolt, Michael; Amenitsch, Heinz; Laggner, Peter

    2000-09-01

    We present a method for analyzing small angle x-ray scattering data on multilamellar phospholipid bilayer systems at full hydration. The method utilizes a modified Caillé theory structure factor in combination with a Gaussian model representation of the electron density profile such that it accounts also for the diffuse scattering between Bragg peaks. Thus the method can retrieve structural information even if only a few orders of diffraction are observed. We further introduce a procedure to derive fundamental parameters, such as area per lipid, membrane thickness, and number of water molecules per lipid, directly from the electron density profile without the need of additional volumetric measurements. The theoretical apparatus is applied to experimental data on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine liposome preparations.

  9. Chimeras of sperm PLCζ reveal disparate protein domain functions in the generation of intracellular Ca2+ oscillations in mammalian eggs at fertilization

    PubMed Central

    Theodoridou, Maria; Nomikos, Michail; Parthimos, Dimitris; Gonzalez-Garcia, J. Raul; Elgmati, Khalil; Calver, Brian L.; Sideratou, Zili; Nounesis, George; Swann, Karl; Lai, F. Anthony

    2013-01-01

    Phospholipase C-zeta (PLCζ) is a sperm-specific protein believed to cause Ca2+ oscillations and egg activation during mammalian fertilization. PLCζ is very similar to the somatic PLCδ1 isoform but is far more potent in mobilizing Ca2+ in eggs. To investigate how discrete protein domains contribute to Ca2+ release, we assessed the function of a series of PLCζ/PLCδ1 chimeras. We examined their ability to cause Ca2+ oscillations in mouse eggs, enzymatic properties using in vitro phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and their binding to PIP2 and PI(3)P with a liposome interaction assay. Most chimeras hydrolyzed PIP2 with no major differences in Ca2+ sensitivity and enzyme kinetics. Insertion of a PH domain or replacement of the PLCζ EF hands domain had no deleterious effect on Ca2+ oscillations. In contrast, replacement of either XY-linker or C2 domain of PLCζ completely abolished Ca2+ releasing activity. Notably, chimeras containing the PLCζ XY-linker bound to PIP2-containing liposomes, while chimeras containing the PLCζ C2 domain exhibited PI(3)P binding. Our data suggest that the EF hands are not solely responsible for the nanomolar Ca2+ sensitivity of PLCζ and that membrane PIP2 binding involves the C2 domain and XY-linker of PLCζ. To investigate the relationship between PLC enzymatic properties and Ca2+ oscillations in eggs, we have developed a mathematical model that incorporates Ca2+-dependent InsP3 generation by the PLC chimeras and their levels of intracellular expression. These numerical simulations can for the first time predict the empirical variability in onset and frequency of Ca2+ oscillatory activity associated with specific PLC variants. PMID:24152875

  10. Herpes simplex virus type 1 and 2 intracellular p40: type-specific and cross-reactive antigenic determinants on peptides generated by partial proteolysis.

    PubMed Central

    Heilman, C J; Zweig, M; Hampar, B

    1981-01-01

    Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants. Images PMID:6172597

  11. Correlation between salt-induced change in water structure and lipid structure of multi-lamellar vesicles observed by terahertz time-domain spectroscopy

    NASA Astrophysics Data System (ADS)

    Choi, Da-Hye; Son, Heyjin; Jeong, Jin-Young; Park, Gun-Sik

    2016-08-01

    Salt-induced change in the structure of water molecules inside multi-lamellar vesicles is experimentally studied with terahertz time-domain spectroscopy. The complex dielectric constant of 1, 2-ditetradecanoyl-sn-glycero-3-phosphocholine model membranes dispersed in NaCl solution with different salt concentrations are measured. Structure of water molecules in the solution is characterized from the measured dielectric constant using Debye relaxation model. Combined with small-angle X-ray scattering observation, it is found that salt-induced change of water structure, especially fast water fraction, show strong correlation with the increase in nanometer-scale multi-lamellar repeat space. This suggests that water could be critical in nanometer-scale membrane-membrane communications.

  12. Microfluidic SAXS Study of Lamellar and Multilamellar Vesicle Phases of Linear Sodium Alkylbenzenesulfonate Surfactant with Intrinsic Isomeric Distribution.

    PubMed

    Poulos, Andreas S; Nania, Manuela; Lapham, Paul; Miller, Ruhina M; Smith, Andrew J; Tantawy, Hossam; Caragay, Joel; Gummel, Jérémie; Ces, Oscar; Robles, Eric S J; Cabral, João T

    2016-06-14

    The structure and flow behavior of a concentrated aqueous solution (45 wt %) of the ubiquitous linear sodium alkylbenzenesulfonate (NaLAS) surfactant is investigated by microfluidic small-angle X-ray scattering (SAXS) at 70 °C. NaLAS is an intrinsically complex mixture of over 20 surfactant molecules, presenting coexisting micellar (L1) and lamellar (Lα) phases. Novel microfluidic devices were fabricated to ensure pressure and thermal resistance, ability to handle viscous fluids, and low SAXS background. Polarized light optical microscopy showed that the NaLAS solution exhibits wall slip in microchannels, with velocity profiles approaching plug flow. Microfluidic SAXS demonstrated the structural spatial heterogeneity of the system with a characteristic length scale of 50 nL. Using a statistical flow-SAXS analysis, we identified the micellar phase and multiple coexisting lamellar phases with a continuous distribution of d spacings between 37.5 and 39.5 Å. Additionally, we showed that the orientation of NaLAS lamellar phases is strongly affected by a single microfluidic constriction. The bilayers align parallel to the velocity field upon entering a constriction and perpendicular to it upon exiting. On the other hand, multilamellar vesicle phases are not affected under the same flow conditions. Our results demonstrate that despite the compositional complexity inherent to NaLAS, microfluidic SAXS can rigorously elucidate its structure and flow response. PMID:27196820

  13. No correlation between multilamellar bodies in the inner ear and further organs of mutant (backstroke, bks) and wildtype zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Anken, R.; Ibsch, M.; Kniesel, U.; Rahmann, H.

    2004-01-01

    The origin of the proteinacious matrix of the inner ear stones (otoliths) of vertebrates has not yet been clarified. Using the backstroke mutant (bks) of the zebrafish Danio rerio, which is characterized by a complete lack of otoliths, we searched for possibly missing or aberrant structural components within the macular epithelia of the inner ears of embryos on the ultrastructural level. Numerous multilamellar bodies (MLBs) were found. The MLBs were, however, not restricted to the inner ears of mutants but were also found in wildtype individuals and in further organs such as brain and liver. MLBs have hitherto never been described from the inner ear of fish and are generally estimated to be rare structures. Their occurrence in fish liver can, however, be induced by using particular chemical substances, which seem to effect adaptive compensatory processes on the cellular level. Such a chemical treatment also affects the ultrastructure of further organelles. Since the occurrence of MLBs in the liver of zebrafish was not accompanied by an alteration of the morphology of other organelles, their occurrence seems not to be due to environmental stress. The findings indicate that the MLBs cannot be correlated with bks-inherent features as well as with missing otolith development/growth. Since the occurrence of MLBs was independent from the developmental stage of a specimen and its overall tissue preservation, it can moreover be excluded that these MLBs merely represent fixation artifacts. Their presence more likely indicates cellular remodelling processes of hitherto unknown significance.

  14. Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium discoideum: social amoebae can also package bacteria.

    PubMed

    Paquet, Valérie E; Charette, Steve J

    2016-03-01

    Many bacteria can resist phagocytic digestion by various protozoa. Some of these bacteria (all human pathogens) are known to be packaged in multilamellar bodies produced in the phagocytic pathway of the protozoa and that are secreted into the extracellular milieu. Packaged bacteria are protected from harsh conditions, and the packaging process is suspected to promote bacterial persistence in the environment. To date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, have been shown to perform bacteria packaging. It is still unknown if social amoebae can do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the grazing of the social amoeba Dictyostelium discoideum and to be packaged by this amoeba was investigated in the present study. The 45 bacterial isolates displaying a resisting phenotype were tested for their capacity to be packaged. A total of seven isolates from Cupriavidus, Micrococcus, Microbacterium and Rathayibacter genera seemed to be packaged and secreted by D. discoideum based on immunofluorescence results. Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were formally packaged. These results show that social amoebae can package some bacteria from the environment revealing a new aspect of microbial ecology. PMID:26862140

  15. Intracellular Zn(2+) Increase in Cardiomyocytes Induces both Electrical and Mechanical Dysfunction in Heart via Endogenous Generation of Reactive Nitrogen Species.

    PubMed

    Tuncay, Erkan; Turan, Belma

    2016-02-01

    Oxidants increase intracellular free Zn(2+) concentration ([Zn(2+)]i) in ventricular myocytes, which contributes to oxidant-induced alterations in excitation-contraction coupling (ECC). However, it is not clear whether increased [Zn(2+)]i in cardiomyocytes via increased reactive nitrogen species (RNS) has a role on heart function under pathological conditions, such as hyperglycemia. In this study, first we aimed to investigate the role of increased [Zn(2+)]i under in vitro condition in the development of both electrical and mechanical dysfunction of isolated papillary muscle strips from rat heart via exposed samples to a Zn(2+)-ionophore (Zn-pyrithione; 1 μM) for 20 min. Under simultaneous measurement of intracellular action potential and contractile activity in these preparations, Zn-pyrithione exposure caused marked prolongation in action potential repolarization phase and slowdown in both contraction and relaxation rates of twitch activity. Second, in order to demonstrate an association between increased [Zn(2+)]i and increased RNS, we monitored intracellular [Zn(2+)]i under an acute exposure of nitric oxide (NO) donor sodium nitroprusside, SNP, in freshly isolated quiescent cardiomyocytes loaded with FluoZin-3. Resting level of free Zn(2+) is significantly higher in cardiomyocytes under hyperglycemic condition compared to those of the controls, which seems to be associated with increased level of RNS production in hyperglycemic cardiomyocytes. Western blot analysis showed that Zn-pyrithione exposure induced a marked decrease in the activity of protein phosphatase 1 and 2A, member of macromolecular protein complex of cardiac ryanodine receptors, RyR2, besides significant increase in the phosphorylation level of extracellular signal-regulated kinase1/2 as a concentration-dependent manner. Overall, the present data demonstrated that there is a cross-relationship between increased RNS production and increased [Zn(2+)]i level in cardiomyocytes under pathological

  16. Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies.

    PubMed

    Reaves, B J; Row, P E; Bright, N A; Luzio, J P; Davidson, H W

    2000-11-01

    A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line. Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway

  17. The Clinical Efficacy of Mometasone Furoate in Multi-Lamellar Emulsion for Eczema: A Double-blinded Crossover Study

    PubMed Central

    Kim, Duk Han; Lee, Hyun Jong; Park, Chun Wook; Kim, Kyu Han; Lee, Kwang Hoon; Ro, Byung In

    2013-01-01

    Background Topical application of corticosteroids also has an influence on skin barrier impairment. Physiological lipid mixtures, such as multi-lamellar emulsion (MLE) containing a natural lipid component leads to effective recovery of the barrier function. Objective The purpose of this study was to conduct an evaluation of the therapeutic efficacy and skin barrier protection of topical mometasone furoate in MLE. Methods A multi-center randomized, double-blind, controlled study was performed to assess the efficacy and safety of mometasone furoate cream in MLE for Korean patients with eczema. The study group included 175 patients with eczema, who applied either mometasone furoate in MLE cream or methylprednisolone aceponate cream for 2 weeks. Treatment efficacy was evaluated using the physician's global assessment of clinical response (PGA), trans-epidermal water loss (TEWL), and visual analogue scale (VAS) for pruritus. Patients were evaluated using these indices at days 4, 8, and 15. Results Comparison of PGA score, TEWL, and VAS score at baseline with those at days 4, 8, and 15 of treatment showed a significant improvement in both groups. Patients who applied mometasone furoate in MLE (74.8%) showed better results (p<0.05) than those who applied methylprednisolone aceponate (47.8%). The TEWL improvement ratio was higher in the mometasone furoate in MLE group than that in the methylprednisolone aceponate group, and VAS improvement was also better in the mometasone furoate in MLE group. Conclusion Mometasone furoate in MLE has a better therapeutic efficacy as well as less skin barrier impairment than methylprednisolone aceponate. PMID:23467551

  18. Allicin protects rat cardiomyoblasts (H9c2 cells) from hydrogen peroxide-induced oxidative injury through inhibiting the generation of intracellular reactive oxygen species.

    PubMed

    Chan, Jackie Yan-Yan; Tsui, Hei-Tung; Chung, Ivan Ying-Ming; Chan, Robbie Yat-Kan; Kwan, Yiu-Wa; Chan, Shun-Wan

    2014-11-01

    Oxidative stress is considered an important factor that promotes cell death in response to a variety of pathophysiological conditions. This study investigated the antioxidant properties of allicin, the principle ingredient of garlic, on preventing oxidative stress-induced injury. The antioxidant capacities of allicin were measured by using 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and hydrogen peroxide (H(2)O(2))-induced cell damage on H9c2 cardiomyoblasts. Allicin (0.3-10 μM) pre-incubation could concentration-dependently attenuate the intracellular reactive oxygen species (ROS) increase induced by H(2)O(2) on H9c2 cells. It could also protect H9c2 cells against H(2)O(2)-induced cell damage. However, the DPPH free radical scavenging activity of allicin was shown to be low. Therefore, it is believed that the protective effect of allicin on H9c2 cells could inhibit intracellular ROS production instead of scavenging extracellular H(2)O(2) or free radicals. For the observed protective effect on H9c2 cells, allicin might also be effective in reducing free radical-induced myocardial cell death in ischemic condition. PMID:24945597

  19. Increased generation of intracellular reactive oxygen species initiates selective cytotoxicity against the MCF-7 cell line resultant from redox active combination therapy using copper-thiosemicarbazone complexes.

    PubMed

    Akladios, Fady N; Andrew, Scott D; Parkinson, Christopher J

    2016-06-01

    The combination of cytotoxic copper-thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity-allowing minimization of the more toxic copper-thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper-thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts. PMID:26951232

  20. Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts.

    PubMed

    Costello, M Joseph; Johnsen, Sönke; Metlapally, Sangeetha; Gilliland, Kurt O; Frame, Lesley; Balasubramanian, Dorairajan

    2010-12-01

    The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as

  1. Intracellular Parasite Invasion Strategies

    NASA Astrophysics Data System (ADS)

    Sibley, L. D.

    2004-04-01

    Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

  2. Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells.

    PubMed Central

    Nezu, Akihiro; Tanimura, Akihiko; Morita, Takao; Irie, Kazuharu; Yajima, Toshihiko; Tojyo, Yosuke

    2002-01-01

    Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells. PMID:11903047

  3. Protective Effects of N-Acetyl Cysteine against Diesel Exhaust Particles-Induced Intracellular ROS Generates Pro-Inflammatory Cytokines to Mediate the Vascular Permeability of Capillary-Like Endothelial Tubes

    PubMed Central

    Tseng, Chia-Yi; Chang, Jing-Fen; Wang, Jhih-Syuan; Chang, Yu-Jung; Gordon, Marion K.; Chao, Ming-Wei

    2015-01-01

    Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascular diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cytokines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione. PMID:26148005

  4. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  5. Global small-angle X-ray scattering data analysis for multilamellar vesicles: the evolution of the scattering density profile model

    PubMed Central

    Heftberger, Peter; Kollmitzer, Benjamin; Heberle, Frederick A.; Pan, Jianjun; Rappolt, Michael; Amenitsch, Heinz; Kučerka, Norbert; Katsaras, John; Pabst, Georg

    2014-01-01

    The highly successful scattering density profile (SDP) model, used to jointly analyze small-angle X-ray and neutron scattering data from unilamellar vesicles, has been adapted for use with data from fully hydrated, liquid crystalline multilamellar vesicles (MLVs). Using a genetic algorithm, this new method is capable of providing high-resolution structural information, as well as determining bilayer elastic bending fluctuations from standalone X-ray data. Structural parameters such as bilayer thickness and area per lipid were determined for a series of saturated and unsaturated lipids, as well as binary mixtures with cholesterol. The results are in good agreement with previously reported SDP data, which used both neutron and X-ray data. The inclusion of deuterated and non-deuterated MLV neutron data in the analysis improved the lipid backbone information but did not improve, within experimental error, the structural data regarding bilayer thickness and area per lipid. PMID:24587787

  6. Astrocytes and oligodendrocytes can be generated from NG2+ progenitors after acute brain injury: intracellular localization of oligodendrocyte transcription factor 2 is associated with their fate choice.

    PubMed

    Zhao, Jing-Wei; Raha-Chowdhury, Ruma; Fawcett, James W; Watts, Colin

    2009-05-01

    Brain injury induces gliosis and scar formation; its principal cell types are mainly astrocytes and some oligodendrocytes. The origin of the astrocytes and oligodendrocytes in the scar remains unclear together with the underlying mechanism of their fate choice. We examined the response of oligodendrocyte transcription factor (Olig)2(+) glial progenitors to acute brain injury. Both focal cortical (mechanical or excitotoxic) and systemic (kainic acid-induced seizure or lipopolysaccharide-induced inflammation) injury caused cytoplasmic translocation of Olig2 (Olig2(TL)) exclusively in affected brain regions as early as 2 h after injury in two-thirds of Olig2(+) cells. Many of the proliferating Olig2(+) cells reacting to injury co-expressed chondroitin sulphate proteoglycan neuron/glia antigen 2 (NG2). Using 5-bromodeoxyuridine (BrdU) tracing protocols, proliferating Olig2(TL)GFAP(+)BrdU(+) cells were observed from 2 days post-lesion (dpl). Immature oligodendrocytes were also seen from 2 dpl and all of them retained Olig2 in the nucleus (Olig2(Nuc)). From 5 dpl Olig2(TL)NG2(+)GFAP(+) cells were observed in the wound and some of them were proliferative. From 5 dpl NG2(+)RIP(+) cells were also seen, all of which were Olig2(Nuc) and some of which were also BrdU(+). Our results suggest that, in response to brain injury, NG2(+) progenitors may generate a subpopulation of astrocytes in addition to oligodendrocytes and their fate choice was associated with Olig2(TL) or Olig2(Nuc). However, the NG2(+)GFAP(+) phenotype was only seen within a limited time window (5-8 dpl) when up to 20% of glial fibrillary acidic protein (GFAP) cells co-expressed NG2. We also observed Olig2(TL)GFAP(+) cells that appeared after injury and before the NG2(+)GFAP(+) phenotype. This suggests that not all astrocytes are derived from an NG2(+) population. PMID:19473238

  7. Managing intracellular transport

    PubMed Central

    Chua, John J.E.; Jahn, Reinhard; Klopfenstein, Dieter R.

    2013-01-01

    Formation and normal function of neuronal synapses are intimately dependent on the delivery to and removal of biological materials from synapses by the intracellular transport machinery. Indeed, defects in intracellular transport contribute to the development and aggravation of neurodegenerative disorders. Despite its importance, regulatory mechanisms underlying this machinery remain poorly defined. We recently uncovered a phosphorylation-regulated mechanism that controls FEZ1-mediated Kinesin-1-based delivery of Stx1 into neuronal axons. Using C. elegans as a model organism to investigate transport defects, we show that FEZ1 mutations resulted in abnormal Stx1 aggregation in neuronal cell bodies and axons. This phenomenon closely resembles transport defects observed in neurodegenerative disorders. Importantly, diminished transport due to mutations of FEZ1 and Kinesin-1 were concomitant with increased accumulation of autophagosomes. Here, we discuss the significance of our findings in a broader context in relation to regulation of Kinesin-mediated transport and neurodegenerative disorders. PMID:24058857

  8. Intracellular Secretory Leukoprotease Inhibitor Modulates Inositol 1,4,5-Triphosphate Generation and Exerts an Anti-Inflammatory Effect on Neutrophils of Individuals with Cystic Fibrosis and Chronic Obstructive Pulmonary Disease

    PubMed Central

    Reeves, Emer P.; Banville, Nessa; Ryan, Dorothy M.; O'Reilly, Niamh; Bergin, David A.; Pohl, Kerstin; Molloy, Kevin; McElvaney, Oliver J.; Alsaleh, Khalifah; Aljorfi, Ahmed; Kandalaft, Osama; O'Flynn, Eimear; Geraghty, Patrick; O'Neill, Shane J.; McElvaney, Noel G.

    2013-01-01

    Secretory leukoprotease inhibitor (SLPI) is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca2+) levels which is mediated by production of inositol 1,4,5-triphosphate (IP3) in response to G-protein-coupled receptor (GPCR) stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n = 10), individuals with cystic fibrosis (CF) (n = 5) or chronic obstructive pulmonary disease (COPD) (n = 5). Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP) and interleukin(IL)-8 induced neutrophil chemotaxis (P < 0.05) and decreased degranulation of matrix metalloprotease-9 (MMP-9), hCAP-18, and myeloperoxidase (MPO) (P < 0.05). The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca2+ flux. The described attenuation of Ca2+ flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca2+ flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD. PMID:24073410

  9. Torilis japonica extract-generated intracellular ROS induces apoptosis by reducing the mitochondrial membrane potential via regulation of the AMPK-p38 MAPK signaling pathway in HCT116 colon cancer.

    PubMed

    Kim, Guen Tae; Lee, Se Hee; Kim, Young Min

    2016-09-01

    Torilis japonica extract (TJE) has been reported to possess diverse medicinal properties including anti‑inflammatory and antibacterial activities. However, the precise mechanism of its anticancer effect is not understood. Thus, we evaluated the apoptotic effects of TJE and examined its underlying molecular mechanisms in HCT116 colorectal cancer cells. Our results show that TJE induces apoptosis through the generation of intracellular reactive oxygen species (ROS), and that it regulates the mitochondrial outer membrane potential via the AMPK/p38 MAPK signaling pathway. Importantly, ~50% of cancer cells have p53 mutations. Thus, the ability to induce apoptosis in a p53-independent manner would be of great value in cancer treatment. Our results show that not only does TJE regulate the AMPK/p38 signaling pathway, but it induces apoptosis in cells in which p53 has been knocked down using siRNA. Moreover, as in in vitro studies, TJE induced apoptosis and regulated apoptosis related-proteins in an HCT 116 xenograft model. Taken together, our results demonstrate that TJE, a natural compound that may provide a substitute for chemotherapeutic drugs, has potential as an anticancer agent. PMID:27314881

  10. Exogenous nitric oxide (NO) generation or IL-1[beta]-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells

    SciTech Connect

    Dimmeler, S.; Bruene, B. ); Ankarcrona, M.; Nicotera, P. )

    1993-04-01

    Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work the authors show that addition of exogenous NO or IL-1[beta]-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 [mu]M of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [[sup 32]P]ADP-ribose from [[sup 32]P]NAD[sup +] in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1[beta] results in decreased GAPDH activity. IL-1[beta] (10[sup [minus]9] M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NAD[sup +] synthease inhibitor N[sub G]-monomethyl-L-arginine, indicating the endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain, NO may mediate autocytotoxic effect in [beta]-cells. 40 refs., 7 figs.

  11. Nanovehicular intracellular delivery systems.

    PubMed

    Prokop, Ales; Davidson, Jeffrey M

    2008-09-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood-brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list "elementary" phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  12. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  13. Topical delivery enhancement with multilamellar liposomes into pilosebaceous units: I. In vitro evaluation using fluorescent techniques with the hamster ear model.

    PubMed

    Lieb, L M; Ramachandran, C; Egbaria, K; Weiner, N

    1992-07-01

    Evidence suggesting liposomal delivery into the pilosebaceous unit of the male Syrian hamster ear membrane was found using two fluorescent techniques, quantitative fluorescence microscopy (QFM), and a scraping method where the various tissue strata of treated skin are analyzed using fluorescence spectrophotometry. Whole ears were mounted on Franz diffusion cells and treated for 24 h with 40 microliters of the following test formulations, each containing approximately 100 micrograms/ml carboxyfluorescein (CF): i) multilamellar phosphatidylcholine: cholesterol: phosphatidylserine liposomes; ii) HEPES buffer (pH, 7.4); iii) 5% propylene glycol; iv) 10% ethanol; v) 0.05% sodium lauryl sulfate; and vi) a suspension of the same lipids used to form the liposomes that were not processed so as to produce a bilayer configuration. Topical application of the liposomally based formulation resulted in a significantly higher accumulation of CF in the pilosebaceous units than the application of any of the other non-liposomal formulations. There was excellent correlation between the two analytical methods used to determine CF deposition into the sebaceous glands. PMID:1607674

  14. Modulation of the multilamellar membrane organization and of the chiral macrodomains in the diatom Phaeodactylum tricornutum revealed by small-angle neutron scattering and circular dichroism spectroscopy.

    PubMed

    Nagy, Gergely; Szabó, Milán; Unnep, Renáta; Káli, György; Miloslavina, Yuliya; Lambrev, Petar H; Zsiros, Ottó; Porcar, Lionel; Timmins, Peter; Rosta, László; Garab, Győző

    2012-03-01

    Diatoms possess effective photoprotection mechanisms, which may involve reorganizations in the photosynthetic machinery. We have shown earlier, by using circular dichroism (CD) spectroscopy, that in Phaeodactylum tricornutum the pigment-protein complexes are arranged into chiral macrodomains, which have been proposed to be associated with the multilamellar organization of the thylakoid membranes and shown to be capable of undergoing light-induced reversible reorganizations (Szabó et al. Photosynth Res 95:237, 2008). Recently, by using small-angle neutron scattering (SANS) on the same algal cells we have determined the repeat distances and revealed reversible light-induced reorganizations in the lamellar order of thylakoids (Nagy et al. Biochem J 436:225, 2011). In this study, we show that in moderately heat-treated samples, the weakening of the lamellar order is accompanied by the diminishment of the psi-type CD signal associated with the long-range chiral order of the chromophores (psi, polymer or salt-induced). Further, we show that the light-induced reversible increase in the psi-type CD is associated with swelling in the membrane system, with magnitudes larger in high light than in low light. In contrast, shrinkage of the membrane system, induced by sorbitol, brings about a decrease in the psi-type CD signal; this shrinkage also diminishes the non-photochemical quenching capability of the cells. These data shed light on the origin of the psi-type CD signal, and confirm that both CD spectroscopy and SANS provide valuable information on the macro-organization of the thylakoid membranes and their dynamic properties; these parameters are evidently of interest with regard to the photoprotection in whole algal cells. PMID:21986933

  15. Intracellular Sterol Dynamics

    PubMed Central

    Mesmin, Bruno; Maxfield, Frederick R.

    2009-01-01

    We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated. PMID:19286471

  16. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  17. High pressure 2H-NMR study of the order and dynamics of selectively deuterated dipalmitoyl phosphatidylcholine in multilamellar aqueous dispersions.

    PubMed Central

    Peng, X.; Jonas, A.; Jonas, J.

    1995-01-01

    High pressure 2H multipulse NMR techniques were used to investigate the effects of pressure on the structure and dynamics of selectively deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilamellar aqueous dispersions. The samples were deuterated on both chains at positions 2, 9, or 13. The deuterium lineshapes, the spin-lattice relaxation times, T1, and the spin-spin relaxation times, T2, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for the three deuterated DPPC samples. This pressure range permitted us to explore the phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as the Gel I (P beta), Gel II (L beta), Gel III, Gel X, and the interdigitated, Gel i, gel phase. Pressure had an ordering effect on all chain segments both in the LC phase and various high pressure gel phases as indicated by the increase in SCD bond order parameter and the first moment, M1, with pressure. Compared with the adjacent gel phases, the Gel i phase had the highest order. Also, in all gel phases the carbon-9 segment of the chains had the most restricted motions in contrast to the LC phase, where the carbon-2 segment was the most restricted. In the LC phase, T1 and T2 values for all segments decreased with pressure, indicative of the fast correlation time regime. Similarly, T1 decreased with pressure in the Gel I and the interdigitated Gel i gel phases but changed to the slow correlation time regime at the Gel i/Gel II phase transition. For T2, which reflects slow motions, the transition to the slow correlation time regime occurred already at LC/Gel I phase transition. Considering the various motions which contribute to relaxation, the behavior of T1 and T2 in the Gel 11 through Gel X phases showing discontinuities and slope changes at the phase transitions was, as expected, quite complex.In addition we found a straight line relationship for T-1 vs. S2D, and T-1 vs. S2CD for the deuterons in the 9

  18. Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging.

    PubMed

    Xu, Jingjing; Huang, Peiyuan; Qin, Yu; Jiang, Dechen; Chen, Hong-Yuan

    2016-05-01

    Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity. PMID:27094779

  19. Generations.

    PubMed

    Chambers, David W

    2005-01-01

    Groups naturally promote their strengths and prefer values and rules that give them an identity and an advantage. This shows up as generational tensions across cohorts who share common experiences, including common elders. Dramatic cultural events in America since 1925 can help create an understanding of the differing value structures of the Silents, the Boomers, Gen Xers, and the Millennials. Differences in how these generations see motivation and values, fundamental reality, relations with others, and work are presented, as are some applications of these differences to the dental profession. PMID:16623137

  20. Decoding of intracellular calcium spike trains

    NASA Astrophysics Data System (ADS)

    Prank, K.; Läer, L.; von zur Mühlen, A.; Brabant, G.; Schöfl, C.

    1998-04-01

    Cells respond to external signals, such as hormonal stimuli, by generating repetitive spikes in the intracellular free-calcium concentration ([Ca2+]i). These [Ca2+]i spikes, which can be modulated in their frequency and amplitude, regulate diverse cellular processes. Experimentally, [Ca2+]i can be assessed continuously in contrast to cellular responses represented by the activation of proteins. We propose a mathematical model that allows for the on-line decoding of [Ca2+]i spike trains into cellular responses represented by the activation of proteins.

  1. Quantitating intracellular oxygen tension in vivo by phosphorescence lifetime measurement

    PubMed Central

    Hirakawa, Yosuke; Yoshihara, Toshitada; Kamiya, Mako; Mimura, Imari; Fujikura, Daichi; Masuda, Tsuyoshi; Kikuchi, Ryohei; Takahashi, Ippei; Urano, Yasuteru; Tobita, Seiji; Nangaku, Masaomi

    2015-01-01

    Hypoxia appears to have an important role in pathological conditions in many organs such as kidney; however, a method to quantify intracellular oxygen tension in vivo has not been well established. In this study, we established an optical method to quantify oxygen tension in mice kidneys using a cationic lipophilic phosphorescence probe, BTPDM1, which has an intracellular oxygen concentration-sensitive phosphorescence lifetime. Since this probe is distributed inside the tubular cells of the mice kidney, we succeeded in detecting acute renal hypoxic conditions and chronic kidney disease. This technique enabled us to estimate intracellular partial pressures of oxygen in vivo by extrapolating the calibration curve generated from cultured tubular cells. Since intracellular oxygen tension is directly related to cellular hypoxic reactions, such as the activation of hypoxia-inducible factors, our method will shed new light on hypoxia research in vivo. PMID:26644023

  2. Intracellular auxin transport in pollen

    PubMed Central

    Dal Bosco, Cristina; Dovzhenko, Alexander; Palme, Klaus

    2012-01-01

    Cellular auxin homeostasis is controlled at many levels that include auxin biosynthesis, auxin metabolism, and auxin transport. In addition to intercellular auxin transport, auxin homeostasis is modulated by auxin flow through the endoplasmic reticulum (ER). PIN5, a member of the auxin efflux facilitators PIN protein family, was the first protein to be characterized as an intracellular auxin transporter. We demonstrated that PIN8, the closest member of the PIN family to PIN5, represents another ER-residing auxin transporter. PIN8 is specifically expressed in the male gametophyte and is located in the ER. By combining genetic, physiological, cellular and biochemical data we demonstrated a role for PIN8 in intracellular auxin homeostasis. Although our investigation shed light on intracellular auxin transport in pollen, the physiological function of PIN8 still remains to be elucidated. Here we discuss our data taking in consideration other recent findings. PMID:22990451

  3. Khz-cp (crude polysaccharide extract obtained from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis by increasing intracellular calcium levels and activating P38 and NADPH oxidase-dependent generation of reactive oxygen species in SNU-1 cells

    PubMed Central

    2014-01-01

    Background Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells. Methods Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes. Results In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. Conclusions In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the

  4. Stochastic models of intracellular calcium signals

    NASA Astrophysics Data System (ADS)

    Rüdiger, Sten

    2014-01-01

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels-one of the most important cellular signaling mechanisms-feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction-diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker-Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  5. ROS and intracellular ion channels.

    PubMed

    Kiselyov, Kirill; Muallem, Shmuel

    2016-08-01

    Oxidative stress is a well-known driver of numerous pathological processes involving protein and lipid peroxidation and DNA damage. The resulting increase of pro-apoptotic pressure drives tissue damage in a host of conditions, including ischemic stroke and reperfusion injury, diabetes, death in acute pancreatitis and neurodegenerative diseases. Somewhat less frequently discussed, but arguably as important, is the signaling function of oxidative stress stemming from the ability of oxidative stress to modulate ion channel activity. The evidence for the modulation of the intracellular ion channels and transporters by oxidative stress is constantly emerging and such evidence suggests new regulatory and pathological circuits that can be explored towards new treatments for diseases in which oxidative stress is an issue. In this review we summarize the current knowledge on the effects of oxidative stress on the intracellular ion channels and transporters and their role in cell function. PMID:26995054

  6. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  7. Intracellular α-Amylase of Streptococcus mutans

    PubMed Central

    Simpson, Christine L.; Russell, Roy R. B.

    1998-01-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  8. Intracellular alpha-amylase of Streptococcus mutans.

    PubMed

    Simpson, C L; Russell, R R

    1998-09-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  9. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  10. Intracellular pH of acid-tolerant ruminal bacteria.

    PubMed Central

    Russell, J B

    1991-01-01

    Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells. PMID:1781695

  11. Fluorescence Ratio Imaging Of Dynamic Intracellular Signals

    NASA Astrophysics Data System (ADS)

    Harootunian, Alec T.; Kao, J. P.; Tsien, Roger Y.

    1989-12-01

    Traditional biochemical assays of cellular messengers require grinding up thousands or millions of cells for each data point. Such destructive measurements use up large amounts of tissue, have poor time resolution, and cannot assess heterogeneity between individual cells or dynamic spatial localizations. Recent technical advances now enable important ionic signals to be continuously imaged inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+. Binding of these ions shifts the fluorescence excitation spectrum of the corresponding indicator. The ratio of excitation amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to nonuniform cell thickness or dye content. By rapidly alternating between the two ion-sensitive excitation wavelengths, a fluorescence microscope equipped with a low-light television camera and digital image processor can produce dynamic images of intracellular messenger levels. In many populations of cells traditionally assumed to be homogeneous, we find that neighboring individual cells can differ enormously in their cytosolic Ca2+ response to agonist stimulation, some ignoring the stimulus, others raising cytosolic Ca2+ transiently, others showing oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of graded inputs; we are investigating the mechanism of their generation using light flashes to generate pulses of intracellular messengers. Spatial gradients of cytosolic Ca t+ within single cells have been observed in embryos during fertilization and development, neurons exposed to electrical or drug stimulation and in cytotoxic T lymphocytes during killing of target

  12. Intracellular targeting with engineered proteins.

    PubMed

    Miersch, Shane; Sidhu, Sachdev S

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  13. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  14. Intracellular ion channels and cancer.

    PubMed

    Leanza, Luigi; Biasutto, Lucia; Managò, Antonella; Gulbins, Erich; Zoratti, Mario; Szabò, Ildikò

    2013-01-01

    Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome, and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K(+) channels (Ca(2+)-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K(+) channel-3 (TASK-3)), Ca(2+) uniporter MCU, Mg(2+)-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC) and the Permeability Transition Pore (MPTP) contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER)-located inositol 1,4,5-trisphosphate (IP3) receptor, the ER-located Ca(2+) depletion sensor STIM1 (stromal interaction molecule 1), a component of the store-operated Ca(2+) channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment. PMID:24027528

  15. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  16. Predicting the intracellular water compartment using artificial neural network analysis.

    PubMed

    Mohamed, E I; Maiolo, C; Linder, R; Pöppl, S J; De Lorenzo, A

    2003-10-01

    Artificial neural networks (ANN) are used for a wide variety of data-processing applications such as predicting medical outcomes and classifying clinical data and patients. We investigated the applicability of an ANN for estimating the intracellular water compartment for a population of 104 healthy Italians ranging in age from 19 to 68 years. Anthropometric variables, bioelectric impedance analysis (BIA) variables, and reference values for intracellular water, measured using whole-body (40)K counting (ICW(K40)), were measured for all study participants. The anthropometric variables and the impedance index (height(2)/resistance) were fed to the ANN input layer, which produced as output the estimated values for intracellular water (ICW(ANN)). We also estimated intracellular water using a BIA formula for the same population (ICW(DeLorenzo)) and another for Caucasians (ICW(Gudivaka)). Errors in the estimations generated by ANN and the BIA equations were calculated as the root mean square error (RMSE). The mean (+/-SD) reference value (ICWK40) was 25.01+/-4.50 l, whereas the mean estimated value was 15.20+/-1.79 l (RMSE=11.06 l) when calculated using ICW(DeLorenzo), 18.07+/-1.14 l (RMSE=8.72 l) when using ICW(Gudivaka), and 25.01+/-2.74 l (RMSE=3.22 l) when using ICW(ANN). Based on these results, we deduce that the ANN algorithm is a more accurate predictor for reference ICW(K40) than BIA equations. PMID:14618426

  17. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  18. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  19. Secretome of obligate intracellular Rickettsia

    PubMed Central

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  20. Dynamics of intracellular information decoding

    NASA Astrophysics Data System (ADS)

    Kobayashi, Tetsuya J.; Kamimura, Atsushi

    2011-10-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

  1. Intracellular signalling during neutrophil recruitment.

    PubMed

    Mócsai, Attila; Walzog, Barbara; Lowell, Clifford A

    2015-08-01

    Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage. PMID:25998986

  2. Intracellular calcium levels as screening tool for nanoparticle toxicity

    PubMed Central

    Meindl, Claudia; Kueznik, Tatjana; Bösch, Martina; Roblegg, Eva; Fröhlich, Eleonore

    2015-01-01

    The use of engineered nano-sized materials led to revolutionary developments in many industrial applications and in the medical field. These materials, however, also may cause cytotoxicity. In addition to size, surface properties and shape were identified as relevant parameters for cell damage. Cell damage may occur as disruption of membrane integrity, induction of apoptosis and by organelle damage. Generation of oxidative stress may serve as an indicator for cytotoxicity. Effects occurring upon short contact of particles with cells, for instance in the systemic blood circulation, could be identified according to increases of intracellular [Ca2+] levels, which are caused by variety of toxic stimuli. Negatively charged, neutral and positively charged polystyrene particles of different sizes were used to study the role of size and surface properties on viability, membrane disruption, apoptosis, lysosome function, intracellular [Ca2+] levels and generation of oxidative stress. Silica particles served to test this hypothesis. Twenty nm polystyrene particles as well as 12 nm and 40 nm silica particles caused membrane damage and apoptosis with no preference of the surface charge. Only 20 nm plain and amine functionalized polystyrene particles cause oxidative stress and only the plain particles lysosomal damage. A potential role of surface charge was identified for 200 nm polystyrene particles, where only the amidine particles caused lysosomal damage. Increases in intracellular [Ca2+] levels and cytotoxicity after 24 h was often linked but determination of intracellular [Ca2+] levels could serve to characterize further the type of membrane damage. © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd. Nano-sized materials may cause cytotoxicity. Negatively charged, neutral and positively charged polystyrene particles of different sizes and silica nanoparticles were used to study the role of size and surface properties on viability, membrane

  3. Intracellular Mechanics of Migrating FibroblastsD⃞

    PubMed Central

    Kole, Thomas P.; Tseng, Yiider; Jiang, Ingjye; Katz, Joseph L.; Wirtz, Denis

    2005-01-01

    Cell migration is a highly coordinated process that occurs through the translation of biochemical signals into specific biomechanical events. The biochemical and structural properties of the proteins involved in cell motility, as well as their subcellular localization, have been studied extensively. However, how these proteins work in concert to generate the mechanical properties required to produce global motility is not well understood. Using intracellular microrheology and a fibroblast scratch-wound assay, we show that cytoskeleton reorganization produced by motility results in mechanical stiffening of both the leading lamella and the perinuclear region of motile cells. This effect is significantly more pronounced in the leading edge, suggesting that the mechanical properties of migrating fibroblasts are spatially coordinated. Disruption of the microtubule network by nocodazole treatment results in the arrest of cell migration and a loss of subcellular mechanical polarization; however, the overall mechanical properties of the cell remain mostly unchanged. Furthermore, we find that activation of Rac and Cdc42 in quiescent fibroblasts elicits mechanical behavior similar to that of migrating cells. We conclude that a polarized mechanics of the cytoskelton is essential for directed cell migration and is coordinated through microtubules. PMID:15483053

  4. Autophagy and checkpoints for intracellular pathogen defense

    PubMed Central

    Paulus, Geraldine L.C.; Xavier, Ramnik J.

    2015-01-01

    Purpose of review Autophagy plays a crucial role in intracellular defense against various pathogens. Xenophagy is a form of selective autophagy that targets intracellular pathogens for degradation. In addition, several related yet distinct intracellular defense responses depend on autophagy-related (ATG) genes. This review gives an overview of these processes, pathogen strategies to subvert them, and their crosstalk with various cell death programs. Recent findings The recruitment of ATG proteins plays a key role in multiple intracellular defense programs, specifically xenophagy, LC3-associated phagocytosis (LAP), and the IFNγ-mediated elimination of pathogens such as Toxoplasma gondii and murine norovirus. Recent progress has revealed methods employed by pathogens to resist these intracellular defense mechanisms and/or persist in spite of them. The intracellular pathogen load can tip the balance between cell survival and cell death. Further, it was recently observed that LAP is indispensable for the efficient clearance of dying cells. Summary Autophagy-dependent and ATG gene-dependent pathways are essential in intracellular defense against a broad range of pathogens. PMID:25394238

  5. Protein-coat dynamics and cluster phases in intracellular trafficking

    NASA Astrophysics Data System (ADS)

    Huber, Greg; Wang, Hui; Mukhopadhyay, Ranjan

    2011-09-01

    Clustering of membrane proteins is a hallmark of biological membranes' lateral organization and crucial to their function. However, the physical properties of these protein aggregates remain poorly understood. Ensembles of coat proteins, the example considered here, are necessary for intracellular transport in eukaryotic cells. Assembly and disassembly rates for coat proteins involved in intracellular vesicular trafficking must be carefully controlled: their assembly deforms the membrane patch and drives vesicle formation, yet the protein coat must rapidly disassemble after vesiculation. Motivated by recent experimental findings for protein-coat dynamics, we study a dynamical Ising-type model for coat assembly and disassembly, and demonstrate how simple dynamical rules generate a robust, steady-state distribution of protein clusters (corresponding to intermediate budded shapes) and how cluster sizes are controlled by the kinetics. We interpret the results in terms of both vesiculation and the coupling to cargo proteins.

  6. Design of biomaterials for intracellular delivery of carbon monoxide.

    PubMed

    Inaba, Hiroshi; Fujita, Kenta; Ueno, Takafumi

    2015-11-01

    Carbon monoxide (CO) is recognized as one of the most important gas signaling molecules involved in governing various therapeutic responses. Intracellular generation of CO is spatiotemporally controlled by catalytic reactions of heme oxygenases (HOs). Thus, the ability to control intracellular CO delivery with modulation of the CO-release rate in specific amounts and locations is expected to improve our fundamental understanding of the functions of CO and the development of clinical applications. For this purpose, CO-releasing molecules (CORMs) have been developed and investigated in vitro and in vivo. Most CORMs are based on transition metal carbonyl complexes. Recently, various biomaterials consisting of metal carbonyls with biomacromolecular scaffolds have been reported to improve the properties of bare metal carbonyls. In this mini-review, current progress in CO delivery, recent strategies for the development of CORMs, and future directions in this field are discussed. PMID:26252321

  7. Stochastic resonance in an intracellular genetic perceptron.

    PubMed

    Bates, Russell; Blyuss, Oleg; Zaikin, Alexey

    2014-03-01

    Intracellular genetic networks are more intelligent than was first assumed due to their ability to learn. One of the manifestations of this intelligence is the ability to learn associations of two stimuli within gene-regulating circuitry: Hebbian-type learning within the cellular life. However, gene expression is an intrinsically noisy process; hence, we investigate the effect of intrinsic and extrinsic noise on this kind of intracellular intelligence. We report a stochastic resonance in an intracellular associative genetic perceptron, a noise-induced phenomenon, which manifests itself in noise-induced increase of response in efficiency after the learning event under the conditions of optimal stochasticity. PMID:24730883

  8. Visualization of Intracellular Tyrosinase Activity in vitro

    PubMed Central

    Setty, Subba Rao Gangi

    2016-01-01

    Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes. PMID:27231711

  9. Supramolecular nanoreactors for intracellular singlet-oxygen sensitization

    NASA Astrophysics Data System (ADS)

    Swaminathan, Subramani; Fowley, Colin; Thapaliya, Ek Raj; McCaughan, Bridgeen; Tang, Sicheng; Fraix, Aurore; Burjor, Captain; Sortino, Salvatore; Callan, John F.; Raymo, Françisco M.

    2015-08-01

    An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.An amphiphilic

  10. Polymer physics of intracellular phase transitions

    NASA Astrophysics Data System (ADS)

    Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.

    2015-11-01

    Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biological roles, by compartmentalizing the cell to enable spatiotemporal control of biological reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liquid droplets that form via phase separation. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liquid-liquid phase separation in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equilibrium features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics.

  11. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Paulo, Cristiana S. O.; Pires das Neves, Ricardo; Ferreira, Lino S.

    2011-12-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  12. Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.

    PubMed

    Aiba, Isamu; West, Adrian K; Sheline, Christian T; Shuttleworth, C William

    2013-06-01

    We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn(2+) accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole-cell clamp revealed a high vulnerability of intracellular Zn(2+) buffers to intracellular dialysis. Thus, low concentrations of zinc-pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation-sensitive Zn(2+) pools was reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein-3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn(2+) influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin-3 signals in wild-type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn(2+) (ZnT3-KO). In contrast, under brief dialysis conditions, intracellular Zn(2+) transients were very similar in WT and ZnT3-KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn(2+) accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn(2+) accumulation mechanisms. PMID:23517525

  13. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    NASA Astrophysics Data System (ADS)

    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  14. Ectdomain shedding and regulated intracellular proteolysis in the central nervous system.

    PubMed

    Montes de Oca-B, Pavel

    2010-12-01

    The term Ectodomain Shedding (ES) refers to extracellular domain proteolytic release from cell membrane molecules. This proteolysis is mediated mainly by matrix metalloproteases (MMP) or disintegrin and metalloproteases (ADAM), although some other proteases may mediate it. Virtually, all functional categories of cell membrane molecules are subject of this kind of proteolysis, for this reason ES is involved in different cellular processes such as proliferation, apoptosis, migration, differentiation or pathologies such as inflammation, cancer and degeneration among others. ES releases membrane molecule's extracellular domain (or ectodomain) to the extracellular milieu where it can play different biological functions. ES of transmembrane molecules also generates membrane attached terminal fragments comprising transmembrane and intracellular domains that enable their additional processing by intracellular proteases known as Regulated Intracellular Proteolysis (RIP). This second proteolytic cleavage delivers molecule's intracellular domain (ICD) that carry out intracellular functions. RIP is mediated by the group of intracellular cleaving proteases (i-CLiPs) that include presenilin from the γ-secretase complex. In the CNS the best well known ES is that of the Amyloid Precursor Protein, although many other membrane molecules expressed by cells of the CNS are also subject to ES and RIP. In this review, these molecules are summarized, and some meaningful examples are highlighted and described. In addition, ES and RIP implications in the context of cell biology are discussed. Finally, some considerations that rise from the study of ES and RIP are formulated in view of the unexpected roles of intracellular fragments. PMID:20868353

  15. Rapid measurements of intracellular calcium using a fluorescence plate reader.

    PubMed

    Lin, K; Sadée, W; Quillan, J M

    1999-02-01

    Intracellular calcium is a universal second messenger that can serve as a broad-based measure of receptor activity. Recent developments in multi-well plate fluorescence readers facilitate measurement of intracellular free-calcium levels and reduce reliance on slower, more cumbersome or expensive data collection methods. In this report, we describe a rapid and sensitive method to assay intracellular calcium ions in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells from multi-well plates using a fluorometer equipped with on-line injectors. We examine the compatibility of visible-light excitable dyes Calcium Green-1 and Oregon Green 488 BAPTA-1. Using this assay, we were able to detect and quantify activity from muscarinic and beta-adrenergic receptors endogenous to HEK293 cells and detect calcium signals generated by activation of Gi-coupled recombinant mu-opioid and dopamine D2L receptors, and the Gs-coupled melanocortin subtype 4 (MC4) receptor. Fluorescence signals, stable in HEK293 cells, required the use of Oregon Green 488 BAPTA-1 and an inhibitor of organic anion transport in CHO cells. Under appropriate conditions, both cell types can be used to collect complete concentration-response data for a variety of receptors (including a recombinant muscarinic M1 receptor expressed in CHO cells) from a single plate of dye-loaded cells. PMID:10023544

  16. Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

    PubMed Central

    Mu, Haibo; Tang, Jiangjiang; Liu, Qianjin; Sun, Chunli; Wang, Tingting; Duan, Jinyou

    2016-01-01

    The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet−visible (UV−vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria. PMID:26728712

  17. Supramolecular nanoreactors for intracellular singlet-oxygen sensitization

    NASA Astrophysics Data System (ADS)

    Swaminathan, Subramani; Fowley, Colin; Thapaliya, Ek Raj; McCaughan, Bridgeen; Tang, Sicheng; Fraix, Aurore; Burjor, Captain; Sortino, Salvatore; Callan, John F.; Raymo, Françisco M.

    2015-08-01

    An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy.An amphiphilic

  18. Supramolecular nanoreactors for intracellular singlet-oxygen sensitization.

    PubMed

    Swaminathan, Subramani; Fowley, Colin; Thapaliya, Ek Raj; McCaughan, Bridgeen; Tang, Sicheng; Fraix, Aurore; Captain, Burjor; Sortino, Salvatore; Callan, John F; Raymo, Françisco M

    2015-09-01

    An amphiphilic polymer with multiple decyl and oligo(ethylene glycol) chains attached to a common poly(methacrylate) backbone assembles into nanoscaled particles in aqueous environments. Hydrophobic anthracene and borondipyrromethene (BODIPY) chromophores can be co-encapsulated within the self-assembling nanoparticles and transported across hydrophilic media. The reversible character of the noncovalent bonds, holding the supramolecular containers together, permits the exchange of their components with fast kinetics in aqueous solution. Incubation of cervical cancer (HeLA) cells with a mixture of two sets of nanoparticles, pre-loaded independently with anthracene or BODIPY chromophores, results in guest scrambling first and then transport of co-entrapped species to the intracellular space. Alternatively, incubation of cells with the two sets of nanocarriers in consecutive steps permits the sequential transport of the anthracene and BODIPY chromophores across the plasma membrane and only then allows their co-encapsulation within the same supramolecular containers. Both mechanisms position the two sets of chromophores with complementary spectral overlap in close proximity to enable the efficient transfer of energy intracellularly from the anthracene donors to the BODIPY acceptors. In the presence of iodine substituents on the BODIPY platform, intersystem crossing follows energy transfer. The resulting triplet state can transfer energy further to molecular oxygen with the concomitant production of singlet oxygen to induce cell mortality. Furthermore, the donor can be excited with two near-infrared photons simultaneously to permit the photoinduced generation of singlet oxygen intracellularly under illumination conditions compatible with applications in vivo. Thus, these supramolecular strategies to control the excitation dynamics of multichromophoric assemblies in the intracellular environment can evolve into valuable protocols for photodynamic therapy. PMID:26238536

  19. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  20. Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

    PubMed Central

    Liszewski, M. Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G.; Fara, Antonella F.; Subias, Marta; Pickering, Matthew C.; Drouet, Christian; Meri, Seppo; Arstila, T. Petteri; Pekkarinen, Pirkka T.; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P.; Kemper, Claudia

    2013-01-01

    Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  1. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

    PubMed

    Liszewski, M Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G; Fara, Antonella F; Subias, Marta; Pickering, Matthew C; Drouet, Christian; Meri, Seppo; Arstila, T Petteri; Pekkarinen, Pirkka T; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P; Kemper, Claudia

    2013-12-12

    Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance. PMID:24315997

  2. GABAAergic stimulation modulates intracellular protein arginine methylation.

    PubMed

    Denman, Robert B; Xie, Wen; Merz, George; Sung, Ying-Ju

    2014-06-20

    Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome. PMID:24793772

  3. Intracellular Signal Modulation by Nanomaterials

    PubMed Central

    Hussain, Salik; Garantziotis, Stavros; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Baeza-Squiban, Armelle; Boland, Sonja

    2016-01-01

    A thorough understanding of the interactions of nanomaterials with biological systems and the resulting activation of signal transduction pathways is essential for the development of safe and consumer friendly nanotechnology. Here we present an overview of signaling pathways induced by nanomaterial exposures and describe the possible correlation of their physicochemical characteristics with biological outcomes. In addition to the hierarchical oxidative stress model and a review of the intrinsic and cell-mediated mechanisms of reactive Oxygen species (ROS) generating capacities of nanomaterials, we also discuss other oxidative stress dependent and independent cellular signaling pathways. Induction of the inflammasome, calcium signaling, and endoplasmic reticulum stress are reviewed. Furthermore, the uptake mechanisms can crucially affect the cytotoxicity of nanomaterials and membrane-dependent signaling pathways can be responsible for cellular effects of nanomaterials. Epigenetic regulation by nanomaterials effects of nanoparticle-protein interactions on cell signaling pathways, and the induction of various cell death modalities by nanomaterials are described. We describe the common trigger mechanisms shared by various nanomaterials to induce cell death pathways and describe the interplay of different modalities in orchestrating the final outcome after nanomaterial exposures. A better understanding of signal modulations induced by nanomaterials is not only essential for the synthesis and design of safer nanomaterials but will also help to discover potential nanomedical applications of these materials. Several biomedical applications based on the different signaling pathways induced by nanomaterials are already proposed and will certainly gain a great deal of attraction in the near future. PMID:24683030

  4. Chemical development of intracellular protein heterodimerizers.

    PubMed

    Erhart, Dominik; Zimmermann, Mirjam; Jacques, Olivier; Wittwer, Matthias B; Ernst, Beat; Constable, Edwin; Zvelebil, Marketa; Beaufils, Florent; Wymann, Matthias P

    2013-04-18

    Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system. PMID:23601644

  5. Macrophage defense mechanisms against intracellular bacteria

    PubMed Central

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  6. Endosomal escape: a bottleneck in intracellular delivery.

    PubMed

    Shete, Harshad K; Prabhu, Rashmi H; Patravale, Vandana B

    2014-01-01

    With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives. Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives. PMID:24730275

  7. Multiplexed imaging of intracellular protein networks.

    PubMed

    Grecco, Hernán E; Imtiaz, Sarah; Zamir, Eli

    2016-08-01

    Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry. PMID:27183498

  8. Peroxisome is a reservoir of intracellular calcium.

    PubMed

    Raychaudhury, Bikramjit; Gupta, Shreedhara; Banerjee, Shouvik; Datta, Salil C

    2006-07-01

    We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca(2+)-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium. PMID:16713100

  9. Intracellular Induction of Listeria monocytogenes actA Expression

    PubMed Central

    Shetron-Rama, Lynne M.; Marquis, Hélène; Bouwer, H. G. Archie; Freitag, Nancy E.

    2002-01-01

    Following entry into the host cytosol, the bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors. The expression of actA, whose protein product is required for L. monocytogenes actin-based intracellular motility, is increased by more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures. Two distinct promoter elements have been reported to regulate actA expression. One promoter is located immediately upstream of actA coding sequences, while the second promoter is contributed by the upstream mpl gene via the generation of an mpl-actA-plcB transcript. A series of L. monocytogenes mutants were constructed to define the contributions of individual promoter elements to actA expression. The intracellular induction of actA expression was found to be dependent upon the actA proximal promoter; the mpl promoter appeared to contribute to the extracellular induction of actA but did not affect intracellular levels of expression. The actA promoter is dependent upon a regulatory factor known as PrfA for transcriptional activation; however, no increase in actA expression was detected following the introduction of a high-affinity PrfA binding site within the actA promoter. The presence of a mutationally activated form of PrfA, known as PrfA*, increased overall actA expression in broth-grown cultures of both wild-type and actA promoter mutant strains, but the levels of induction observed were still approximately 50-fold lower than those observed for intracellularly grown L. monocytogenes. Collectively, these results indicate that the dramatic induction of actA expression that occurs in the host cell cytosol is mediated through a single promoter element. Furthermore, intracellular induction of actA appears to require additional steps or factors beyond those necessary for the activation and binding of PrfA to the actA promoter. PMID:11854187

  10. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis.

    PubMed

    Garfoot, Andrew L; Rappleye, Chad A

    2016-02-01

    The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages. PMID:26235362

  11. Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections

    PubMed Central

    Hodgson, Kelly; Morris, Jodie; Bridson, Tahnee; Govan, Brenda; Rush, Catherine; Ketheesan, Natkunam

    2015-01-01

    Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host–pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low-grade inflammation due to activation of pro-inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon-γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen-presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T-cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T-cell-mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper-inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research. PMID:25262977

  12. Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level.

    PubMed

    Salánki, J; Budai, D; Véró, M

    1983-01-01

    The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation. PMID:6198869

  13. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  14. Intracellular electric fields produced by dielectric barrier discharge treatment of skin

    NASA Astrophysics Data System (ADS)

    Babaeva, Natalia Yu; Kushner, Mark J.

    2010-05-01

    The application of atmospheric pressure plasmas to human tissue has been shown to have therapeutic effects for wound healing and in treatment of skin diseases. These effects are attributed to both production of beneficial radicals which intersect with biological reaction chains and to the surface and intracellular generation of electric fields. In this paper, we report on computational studies of the intersection of plasma streamers in atmospheric pressure dielectric barrier discharges (DBDs) sustained in air with human skin tissue, with emphasis on the intracellular generation of electric fields. Intracellular structures and their electrical properties were incorporated into the computational mesh in order to self-consistently couple gas phase plasma transport with the charging of the surface of the skin and the intracellular production of electrical currents. The short duration of a single plasma filament in DBDs and its intersection with skin enables the intracellular penetration of electric fields. The magnitude of these electric fields can reach 100 kV cm-1 which may exceed the threshold for electroporation.

  15. The meteoric rise of regulated intracellular proteolysis.

    PubMed

    Mayer, R J

    2000-11-01

    It is often the case in biology that research into breaking things down lags behind research into synthesizing them, and this is certainly true for intracellular proteolysis. Now that we recognize that intracellular proteolysis, triggered by attaching multiple copies of a small protein called ubiquitin to target proteins, is fundamental to life, it is hard to believe that 20 years ago this field was little more than a backwater of biochemistry studied by a handful of laboratories. Among the few were Avram Hershko, Aaron Ciechanover and Alexander Varshavsky, who were recently awarded the Albert Lasker award for basic medical research for discovering the importance of protein degradation in cellular physiology. This Timeline traces how they and their collaborators triggered the rapid movement of ubiquitin-mediated proteolysis to centre stage. PMID:11253367

  16. The intracellular dynamic of protein palmitoylation

    PubMed Central

    Salaun, Christine; Greaves, Jennifer

    2010-01-01

    S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation. PMID:21187327

  17. A practical approach for intracellular protein delivery

    PubMed Central

    Biri, Stéphanie; Adib, Abdennaji; Erbacher, Patrick

    2007-01-01

    Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery. PMID:19002840

  18. Dynamics of gradient formation by intracellular shuttling

    NASA Astrophysics Data System (ADS)

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-01

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  19. Dynamics of gradient formation by intracellular shuttling

    SciTech Connect

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  20. Toward Intracellular Targeted Delivery of Cancer Therapeutics

    PubMed Central

    Pandya, Hetal; Debinski, Waldemar

    2013-01-01

    A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells. PMID:22671766

  1. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy.

    PubMed

    Cheng, Wan-Yun; Larson, Jeffrey M; Samet, James M

    2014-03-15

    There is increasing interest in using live cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of fluorescent signals from multiple probes is that emission spectra of many fluorophores overlap, often with maxima that are only a few nanometers apart. Spectral acquisition of mixed fluorescence signals captured within a dedicated scanning range can be used to quantitatively separate signals into component spectra. We report here the development of a novel live cell application of spectral unmixing for the simultaneous monitoring of intracellular events reported by closely-emitting fluorophores responding dynamically to external stimuli. We validate the performance of dynamic spectral unmixing microscopy (DynSUM) using genetically encoded sensors to simultaneously monitor changes in glutathione redox potential (Egsh) and H2O2 production in living cells exposed to oxidizing and reducing agents. We further demonstrate the utility of the DynSUM approach to observe the relationship between the increases in Egsh and H2O2 generation induced in airway epithelial cells exposed to an environmental electrophile. PMID:23816786

  2. Transient light-induced intracellular oxidation revealed by redox biosensor

    SciTech Connect

    Kolossov, Vladimir L.; Beaudoin, Jessica N.; Hanafin, William P.; DiLiberto, Stephen J.; Kenis, Paul J.A.; Rex Gaskins, H.

    2013-10-04

    Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.

  3. Intracellular iron concentration of neurons with and without perineuronal nets

    NASA Astrophysics Data System (ADS)

    Fiedler, Anja; Reinert, Tilo; Morawski, Markus; Brückner, Gert; Arendt, Thomas; Butz, Tilman

    2007-07-01

    Neurodegenerative diseases like Parkinson's disease, Alzheimer's disease and Huntington's disease are characterized by abnormally high concentrations of iron in the affected brain areas. Iron is believed to contribute to oxidative stress by catalysing radical generation and subsequently causing neuronal death. Interestingly, subpopulations of neurons are less vulnerable against degeneration. One of these subpopulations possesses a specialized extracellular matrix arranged as a perineuronal net (PN), a structure with poorly understood functions. In order to differentiate between neurons with and without PN according to their iron concentrations we have performed a μPIXE study at the Leipzig LIPSION laboratory. PN-ensheathed neurons in selected brain areas were detected by lectin-histochemical staining with Wisteria floribunda agglutinin (WFA). The staining was intensified by DAB- nickel by an established method enabling the visualisation of the PNs by nuclear microscopy. The cellular concentration of iron in the rat brain was about 1 mmol/l (ca. 30 μg/g dw). First results of subcellular analysis showed that the intracellular iron concentration of PN-ensheathed neurons tends to be slightly increased in comparison to neurons without PNs. The difference in intracellular iron concentrations could be an effect of the PNs.

  4. A bacteriophage endolysin that eliminates intracellular streptococci

    PubMed Central

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  5. Error Propagation Analysis for Quantitative Intracellular Metabolomics

    PubMed Central

    Tillack, Jana; Paczia, Nicole; Nöh, Katharina; Wiechert, Wolfgang; Noack, Stephan

    2012-01-01

    Model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. For quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. The total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. Here, we present a framework for the quantification of intracellular metabolites, including error propagation during metabolome sample processing. Focusing on one specific protocol, we comprehensively investigate all currently known and accessible factors that ultimately impact the accuracy of intracellular metabolite concentration data. All intermediate steps are modeled, and their uncertainty with respect to the final concentration data is rigorously quantified. Finally, on the basis of a comprehensive metabolome dataset of Corynebacterium glutamicum, an integrated error propagation analysis for all parts of the model is conducted, and the most critical steps for intracellular metabolite quantification are detected. PMID:24957773

  6. Intracellular Pressure Dynamics in Blebbing Cells.

    PubMed

    Strychalski, Wanda; Guy, Robert D

    2016-03-01

    Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

  7. A bacteriophage endolysin that eliminates intracellular streptococci.

    PubMed

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. PMID:26978792

  8. Dual responsive nanogels for intracellular doxorubicin delivery.

    PubMed

    Asadi, Hamed; Khoee, Sepideh

    2016-09-10

    Nanosized polymeric delivery systems that encapsulate drug molecules and release them in response to a specific intracellular stimulus are of promising interest for cancer therapy. Here, we demonstrated a simple and fast synthetic protocol of redox-responsive nanogels with high drug encapsulation efficiency and stability. The prepared nanogels displayed narrow size distributions and versatility of surface modification. The polymer precursor of these nanogels is based on a random copolymer that contains oligoethyleneglycol (OEG) and pyridyldisulfide (PDS) units as side-chain functionalities. The nanogels were prepared through a lock-in strategy in aqueous media via self cross-linking of PDS groups. By changing polymer concentration, we could control the size of nanogels in range of 80-115nm. The formed nanogels presented high doxorubicin (DOX) encapsulation efficiency (70% (w/w)) and displayed pH and redox-controlled drug release triggered by conditions mimicking the reducible intracellular environment. The nanogels displayed an excellent cytocompatibility and were effectively endocytosed by A2780CP ovarian cancer cells, which make them promising nanomaterials for the efficient intracellular delivery of anticancer drugs. PMID:27444549

  9. Invasion and Intracellular Survival by Protozoan Parasites

    PubMed Central

    Sibley, L. David

    2013-01-01

    Summary Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a nonfusigenic vacuole (e.g. Toxoplasma, Encephalitizoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunological responses and thereby prevent disease. PMID:21349087

  10. Probing Ordered Lipid Assemblies with Polarized Third-Harmonic-Generation Microscopy

    NASA Astrophysics Data System (ADS)

    Zimmerley, Maxwell; Mahou, Pierre; Débarre, Delphine; Schanne-Klein, Marie-Claire; Beaurepaire, Emmanuel

    2013-01-01

    Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder.

  11. Two complementary approaches for intracellular delivery of exogenous enzymes

    PubMed Central

    Rust, Aleksander; Hassan, Hazirah H. A.; Sedelnikova, Svetlana; Niranjan, Dhevahi; Hautbergue, Guillaume; Abbas, Shaymaa A.; Partridge, Lynda; Rice, David; Binz, Thomas; Davletov, Bazbek

    2015-01-01

    Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes. PMID:26207613

  12. Two complementary approaches for intracellular delivery of exogenous enzymes.

    PubMed

    Rust, Aleksander; Hassan, Hazirah H A; Sedelnikova, Svetlana; Niranjan, Dhevahi; Hautbergue, Guillaume; Abbas, Shaymaa A; Partridge, Lynda; Rice, David; Binz, Thomas; Davletov, Bazbek

    2015-01-01

    Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes. PMID:26207613

  13. Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

    PubMed Central

    García, Isabel; Pascual, Alvaro; Ballesta, Sofía; Joyanes, Providencia; Perea, Evelio J.

    2000-01-01

    The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus. PMID:11036051

  14. Physical constraints on the establishment of intracellular spatial gradients in bacteria

    PubMed Central

    2012-01-01

    Background Bacteria dynamically regulate their intricate intracellular organization involving proteins that facilitate cell division, motility, and numerous other processes. Consistent with this sophisticated organization, bacteria are able to create asymmetries and spatial gradients of proteins by localizing signaling pathway components. We use mathematical modeling to investigate the biochemical and physical constraints on the generation of intracellular gradients by the asymmetric localization of a source and a sink. Results We present a systematic computational analysis of the effects of other regulatory mechanisms, such as synthesis, degradation, saturation, and cell growth. We also demonstrate that gradients can be established in a variety of bacterial morphologies such as rods, crescents, spheres, branched and constricted cells. Conclusions Taken together, these results suggest that gradients are a robust and potentially common mechanism for providing intracellular spatial cues. PMID:22931750

  15. Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

    NASA Astrophysics Data System (ADS)

    Oelstrom, Kevin; Goldschen-Ohm, Marcel P.; Holmgren, Miguel; Chanda, Baron

    2014-03-01

    Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

  16. Structural responses of cells to intracellular magnetic force induced by superparamagnetic iron oxide nanoparticles

    PubMed Central

    Shen, Han; Tong, Sheng; Bao, Gang; Wang, Biao

    2014-01-01

    In this paper, we study the effects of intracellular force on human umbilical vein endothelial cells. We generated intracellular force on endothelial cells under different magnetic fields using the cell uptake of superparamagnetic iron oxide nanoparticles. Cell responses to intracellular force were observed using fluorescent microscopy. Our results indicated that nanoparticles were taken up by the cell by endocytosis and were deposited in lysosomes. Nanoparticles and lysosomes inside the cell could be relocated by the application of a magnetic force. The intracellular magnetic force could also be used to accelerate cell migration by adjusting the magnetic fields and giving the cell free culture space. No cytotoxicity of nanoparticles was found in our experiments. By comparing intracellular relocalization with migration of the whole cell, we obtained a better understanding of the self-defence mechanisms of cells based on their mechanical properties. Based on the promising mechanical properties and low cytotoxicity of our magnetic nanoparticles, their potential applications in cytomechanics and cell patterning are discussed. PMID:24336693

  17. Chemokine receptor internalization and intracellular trafficking.

    PubMed

    Neel, Nicole F; Schutyser, Evemie; Sai, Jiqing; Fan, Guo-Huang; Richmond, Ann

    2005-12-01

    The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors. PMID:15998596

  18. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

    NASA Astrophysics Data System (ADS)

    Betzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.

    2006-09-01

    We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

  19. Kinesin superfamily motor proteins and intracellular transport.

    PubMed

    Hirokawa, Nobutaka; Noda, Yasuko; Tanaka, Yosuke; Niwa, Shinsuke

    2009-10-01

    Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research. PMID:19773780

  20. Macrophage cell death upon intracellular bacterial infection

    PubMed Central

    Lai, Xin-He; Xu, Yunsheng; Chen, Xiao-Ming; Ren, Yi

    2015-01-01

    Macrophage-pathogen interaction is a complex process and the outcome of this tag-of-war for both sides is to live or die. Without attempting to be comprehensive, this review will discuss the complexity and significance of the interaction outcomes between macrophages and some facultative intracellular bacterial pathogens as exemplified by Francisella, Salmonella, Shigella and Yersinia. Upon bacterial infection, macrophages can die by a variety of ways, such as apoptosis, autophagic cell death, necrosis, necroptosis, oncosis, pyronecrosis, pyroptosis etc, which is the focus of this review. PMID:26690967

  1. Intercellular and intracellular functions of ceramides and their metabolites in skin (Review).

    PubMed

    Cha, Hwa Jun; He, Congfen; Zhao, Hua; Dong, Yinmao; An, In-Sook; An, Sungkwan

    2016-07-01

    The skin consists of the epidermis, dermis and subcutis. The epidermis is primarily comprised of keratinocytes and is separated into four layers according to the stage of differentiation of the keratinocytes. Corneocytes are terminally differentiated keratinocytes that closely interact with other corneocytes through corneodesmosomes, and synthesize lamellar bodies and the intercellular multilamellar barrier, which protects the body from the external environment. As ceramides are the principal components of lamellar bodies and the multilamellar barrier, it is important to understand the biosynthesis of ceramides and their functions in skin. Ceramides are synthesized by amide bond‑mediated interactions between sphingoid bases, long‑chain amino alcohols [long-chain base] and fatty acids through a de novo pathway, a sphingomyelin (SM) hydrolysis pathway and a catabolic pathway. The majority of ceramides produced by the de novo pathway form the epidermal barrier. Ceramides used as signaling molecules are synthesized by the SM and catabolic pathways. Synthesized ceramides are released from corneocytes and form the multilamellar barrier. Additionally, ceramides and their metabolites regulate the apoptosis, proliferation and differentiation of skin cells as well as the formation of the skin barrier. Thus, the study of ceramides and their metabolites is crucial to understanding the function and regulation of the skin barrier. PMID:27222347

  2. Intracellular membrane traffic at high resolution

    PubMed Central

    van Weering, Jan R.T.; Brown, Edward; Sharp, Thomas H.; Mantell, Judith; Cullen, Peter J.

    2014-01-01

    I. Abstract Membrane traffic between organelles is essential for a multitude of processes that maintain cell homeostasis. Many steps in these tightly regulated trafficking pathways take place in microdomains on the membranes of organelles, which require analysis at nanometer resolution. Electron Microscopy (EM) can visualize these processes in detail and is mainly responsible for our current view of morphology on the subcellular level. This review discusses how EM can be applied to solve many questions of intracellular membrane traffic, with a focus on the endosomal system. We describe the expansion of the technique from purely morphological analysis to cryo-immuno-EM, Correlative Light Electron Microscopy (CLEM) and 3D electron tomography. In this review we go into some technical details of these various techniques. Furthermore, we provide a full protocol for immunolabeling on Lowicryl sections of high-pressure frozen cells as well as a detailed description of a simple CLEM method that can be applied to answer many membrane trafficking questions. We believe that these EM-based techniques are important tools to expand our understanding of the molecular details of endosomal sorting and intracellular membrane traffic in general. PMID:20869541

  3. Strategies for Intracellular Survival of Burkholderia pseudomallei.

    PubMed

    Allwood, Elizabeth M; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed. PMID:22007185

  4. Small Peptide Recognition Sequence for Intracellular Sorting

    PubMed Central

    Pandey, Kailash N.

    2010-01-01

    Increasing evidence indicate that complex arrays of short signals and recognition peptide sequence ensure accurate trafficking and distribution of transmembrane receptors and/or proteins and their ligands into intracellular compartments. Internalization and subsequent trafficking of cell-surface receptors into the cell interior is mediated by specific short-sequence peptide signals within the cytoplasmic domains of these receptor proteins. The short signals usually consist of small linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. In recent years, much has been learned about the function and mechanisms of endocytic pathways responsible for the trafficking and molecular sorting of membrane receptors and their ligands into intracellular compartments, however, the significance and scope of the short sequence motifs in these cellular events is not well understood. Here a particular emphasis has been given to the functions of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into subcellular compartments. PMID:20817434

  5. Intracellular Calcium Dysregulation: Implications for Alzheimer's Disease

    PubMed Central

    Magi, Simona; Castaldo, Pasqualina; Macrì, Maria Loredana; Maiolino, Marta; Matteucci, Alessandra; Bastioli, Guendalina; Gratteri, Santo; Lariccia, Vincenzo

    2016-01-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level. PMID:27340665

  6. Mucolipins: Intracellular TRPML1-3 Channels

    PubMed Central

    Cheng, Xiping; Shen, Dongbiao; Samie, Mohammad; Xu, Haoxing

    2010-01-01

    The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously over-expressed have revealed the channel properties of TRPMLs in mediating cation (Ca2+/Fe2+) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration. PMID:20074572

  7. Intracellular accumulation of ethanol in yeast

    SciTech Connect

    Loueiro, V.; Ferreira, H.G.

    1983-09-01

    Ethanol produced in the course of a batch fermentation by Saccharomyces cerevisiae or added from the outside, affects adversely the specific rate of growth of the yeast population, its viability, its specific rate of fermentation, and the specific rates of the uptake of sugar and amino acids. The underlying mechanisms are many and include irreversible denaturation and hyperbolic noncompetitive inhibition of glycolytic enzymes, the exponential noncompetitive inhibition of glucose, maltose, and ammonium transport, the depression of the optimum and the maximum temperature for growth, the increase of the minimum temperature for growth, and the enhancement of thermal death and petite mutation. Nagodawithana and Steinkraus reported that added ethanol was less toxic for S. cerevisiae than ethanol produced by the yeast. The death rates were lower in the presence of added ethanol than those measured at similar external ethanol concentrations endogenously produced. They proposed that, due to an unbalance between the rates of production and the net outflux of ethanol, there would be an intracellular accumulation of ethanol which in turn would explain the apparently greater inhibitory potency of endogenously produced ethanol present in the medium. This hypothesis was supported by the findings of several authors who reported that the intracellular concentration of ethanol, in the course of batch fermentation, is much higher than its concentration in the extracellular medium. The present work is an attempt to clarify this matter. (Refs. 32).

  8. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  9. Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages

    PubMed Central

    Thompson, J. A.; Proença, J. T.; Gordo, I.

    2016-01-01

    The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10−6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects. PMID:26752723

  10. Functional characterization of intracellular Dictyostelium discoideum P2X receptors.

    PubMed

    Ludlow, Melanie J; Durai, Latha; Ennion, Steven J

    2009-12-11

    Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. beta,gamma-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na(+) but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl(-)-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling. PMID:19833731

  11. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    PubMed

    Parker, William H; Qu, Zhi-chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  12. Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

    NASA Astrophysics Data System (ADS)

    Arellano, Carlos Luis

    Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

  13. Glycosaminoglycans: Sorting determinants in intracellular protein traffic.

    PubMed

    Mihov, Deyan; Spiess, Martin

    2015-11-01

    Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence or in a protein's post-translational modifications. Glycosaminoglycans (GAGs) are characteristic modifications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and functional properties also contributing to protein sorting in various ways. By deletion or insertion of GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein, was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and proteoglycan trafficking. PMID:26327396

  14. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  15. Impaired intracellular trafficking defines early Parkinson's disease

    PubMed Central

    Hunn, Benjamin H.M.; Cragg, Stephanie J.; Bolam, J. Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

    2015-01-01

    Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment. PMID:25639775

  16. Intracellular signalling by C-peptide.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2008-01-01

    C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na(+)/K(+) ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. PMID:18382618

  17. Intracellular dynamics with the phase microscope Airyscan

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.; Perevedentseva, Elena V.; Vyshenskaia, Tatiana V.; Kufal, Georgy E.

    1997-12-01

    Investigation of intracellular dynamics of Allium cepa inner epidermal cells are described. The applicability of the method for quantitative estimation of spatio-temporal phase fluctuations and the effect due to external factors is discussed. The analysis of time-sampled series allows one to detect the regions of various motility in cytoplasm. The intense Fourier-spectra harmonics in 0.2 - 8 Hz interval were observed inside a cell wall and cytoplasm. Regularly spaced 2- to 4-s long batches of 100-ms pulses at cell-wall sites are recorded. The phase-fluctuation intensity decreased and the frequencies of certain harmonics were shifted with lowering temperature. The advantages and specific features of the method are discussed.

  18. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  19. Cell-targeting aptamers act as intracellular delivery vehicles.

    PubMed

    Gopinath, Subash C B; Lakshmipriya, Thangavel; Chen, Yeng; Arshad, M K Md; Kerishnan, Jesinda P; Ruslinda, A R; Al-Douri, Yarub; Voon, C H; Hashim, Uda

    2016-08-01

    Aptamers are single-stranded nucleic acids or peptides identified from a randomized combinatorial library through specific interaction with the target of interest. Targets can be of any size, from small molecules to whole cells, attesting to the versatility of aptamers for binding a wide range of targets. Aptamers show drug properties that are analogous to antibodies, with high specificity and affinity to their target molecules. Aptamers can penetrate disease-causing microbial and mammalian cells. Generated aptamers that target surface biomarkers act as cell-targeting agents and intracellular delivery vehicles. Within this context, the "cell-internalizing aptamers" are widely investigated via the process of cell uptake with selective binding during in vivo systematic evolution of ligands by exponential enrichment (SELEX) or by cell-internalization SELEX, which targets cell surface antigens to be receptors. These internalizing aptamers are highly preferable for the localization and functional analyses of multiple targets. In this overview, we discuss the ways by which internalizing aptamers are generated and their successful applications. Furthermore, theranostic approaches featuring cell-internalized aptamers are discussed with the purpose of analyzing and diagnosing disease-causing pathogens. PMID:27350620

  20. Imaging intracellular protein dynamics by spinning disk confocal microscopy

    PubMed Central

    Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

    2012-01-01

    The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

  1. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    PubMed Central

    Leon-Sicairos, Nidia; Reyes-Cortes, Ruth; Guadrón-Llanos, Alma M.; Madueña-Molina, Jesús; Leon-Sicairos, Claudia; Canizalez-Román, Adrian

    2015-01-01

    Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed. PMID:26120582

  2. Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators.

    PubMed

    Akam, Eman A; Chang, Tsuhen M; Astashkin, Andrei V; Tomat, Elisa

    2014-10-01

    Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species. PMID:25100578

  3. Quantitative Intracellular Localization of Cationic Lipid-Nucleic Acid Nanoparticles with Fluorescence Microscopy.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-01-01

    Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)-NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid-NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL-NA NPs using quantitative analysis with fluorescence microscopy.The first method, spatial localization, describes how to prepare fluorescently labeled CL-NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP-endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL-NA NPs which can guide their design and improve their efficiency. PMID:27436314

  4. On-demand intracellular amplification of chemoradiation with cancer-specific plasmonic nanobubbles

    PubMed Central

    Lukianova-Hleb, Ekaterina Y; Wu, Xiangwei; Torchilin, Vladimir P; Lapotko, Dmitri O

    2014-01-01

    Chemoradiation-resistant cancers limit treatment efficacy and safety. We show here the cancer cell–specific, on-demand intracellular amplification of chemotherapy and chemoradiation therapy via gold nanoparticle– and laser pulse–induced mechanical intracellular impact. Cancer aggressiveness promotes the clustering of drug nanocarriers and gold nanoparticles in cancer cells. This cluster, upon exposure to a laser pulse, generates a plasmonic nanobubble, the mechanical explosion that destroys the host cancer cell or ejects the drug into its cytoplasm by disrupting the liposome and endosome. The same cluster locally amplifies external X-rays. Intracellular synergy of the mechanical impact of plasmonic nanobubble, ejected drug and amplified X-rays improves the efficacy of standard chemoradiation in resistant and aggressive head and neck cancer by 100-fold in vitro and 17-fold in vivo, reduces the effective entry doses of drugs and X-rays to 2–6% of their clinical doses and efficiently spares normal cells. The developed quadrapeutics technology combines four clinically validated components and transforms a standard macrotherapy into an intracellular on-demand theranostic microtreatment with radically amplified therapeutic efficacy and specificity. PMID:24880615

  5. On-demand intracellular amplification of chemoradiation with cancer-specific plasmonic nanobubbles.

    PubMed

    Lukianova-Hleb, Ekaterina Y; Ren, Xiaoyang; Sawant, Rupa R; Wu, Xiangwei; Torchilin, Vladimir P; Lapotko, Dmitri O

    2014-07-01

    Chemoradiation-resistant cancers limit treatment efficacy and safety. We show here the cancer cell-specific, on-demand intracellular amplification of chemotherapy and chemoradiation therapy via gold nanoparticle- and laser pulse-induced mechanical intracellular impact. Cancer aggressiveness promotes the clustering of drug nanocarriers and gold nanoparticles in cancer cells. This cluster, upon exposure to a laser pulse, generates a plasmonic nanobubble, the mechanical explosion that destroys the host cancer cell or ejects the drug into its cytoplasm by disrupting the liposome and endosome. The same cluster locally amplifies external X-rays. Intracellular synergy of the mechanical impact of plasmonic nanobubble, ejected drug and amplified X-rays improves the efficacy of standard chemoradiation in resistant and aggressive head and neck cancer by 100-fold in vitro and 17-fold in vivo, reduces the effective entry doses of drugs and X-rays to 2-6% of their clinical doses and efficiently spares normal cells. The developed quadrapeutics technology combines four clinically validated components and transforms a standard macrotherapy into an intracellular on-demand theranostic microtreatment with radically amplified therapeutic efficacy and specificity. PMID:24880615

  6. Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

    SciTech Connect

    Selmi, Anna; Malinowski, Mariusz; Brutkowski, Wojciech; Bednarek, Radoslaw; Cierniewski, Czeslaw S.

    2012-08-15

    Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.

  7. Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators

    PubMed Central

    Akam, Eman A.; Chang, Tsuhen M.; Astashkin, Andrei V.

    2014-01-01

    Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species. PMID:25100578

  8. Spatially coordinated changes in intracellular rheology and extracellular force exertion during mesenchymal stem cell differentiation

    NASA Astrophysics Data System (ADS)

    McAndrews, Kathleen M.; McGrail, Daniel J.; Quach, Nhat D.; Dawson, Michelle R.

    2014-10-01

    The mechanical properties within the cell are regulated by the organization of the actin cytoskeleton, which is linked to the extracellular environment through focal adhesion proteins that transmit force. Chemical and mechanical stimuli alter the organization of cytoskeletal actin, which results in changes in cell shape, adhesion, and differentiation. By combining particle-tracking microrheology and traction force cytometry, we can monitor the mechanical properties of the actin meshwork and determine how changes in the intracellular network contribute to force generation. In this study, we investigated the effects of chemical (differentiation factors) and mechanical (substrate rigidity) stimuli important in mesenchymal stem cell (MSC) differentiation on the intracellular mechanics and traction stress generation. We found the presence of adipogenic factors resulted in stiffening of the actin meshwork regardless of substrate rigidity. In contrast, these factors increased traction stresses on hard substrates, which was associated with increased expression of contractility genes. Furthermore, MSCs cultured on hard substrates expressed both adipogenic and osteogenic markers indicative of mixed differentiation. On hard substrates, heterogeneity in the local elastic modulus-traction stress correlation was also increased in response to adipogenic factors, indicating that these mechanical properties may be reflective of differences in the level of MSC differentiation. These results suggest intracellular rheology and traction stress generation are spatially regulated and contribute insight into how single cell mechanical forces contribute to MSC differentiation.

  9. ZnO Nanostructure-Based Intracellular Sensor.

    PubMed

    Asif, Muhammad H; Danielsson, Bengt; Willander, Magnus

    2015-01-01

    Recently ZnO has attracted much interest because of its usefulness for intracellular measurements of biochemical species by using its semiconducting, electrochemical, catalytic properties and for being biosafe and biocompatible. ZnO thus has a wide range of applications in optoelectronics, intracellular nanosensors, transducers, energy conversion and medical sciences. This review relates specifically to intracellular electrochemical (glucose and free metal ion) biosensors based on functionalized zinc oxide nanowires/nanorods. For intracellular measurements, the ZnO nanowires/nanorods were grown on the tip of a borosilicate glass capillary (0.7 µm in diameter) and functionalized with membranes or enzymes to produce intracellular selective metal ion or glucose sensors. Successful intracellular measurements were carried out using ZnO nanowires/nanorods grown on small tips for glucose and free metal ions using two types of cells, human fat cells and frog oocytes. The sensors in this study were used to detect real-time changes of metal ions and glucose across human fat cells and frog cells using changes in the electrochemical potential at the interface of the intracellular micro-environment. Such devices are helpful in explaining various intracellular processes involving ions and glucose. PMID:26007730

  10. ZnO Nanostructure-Based Intracellular Sensor

    PubMed Central

    Asif, Muhammad H.; Danielsson, Bengt; Willander, Magnus

    2015-01-01

    Recently ZnO has attracted much interest because of its usefulness for intracellular measurements of biochemical species by using its semiconducting, electrochemical, catalytic properties and for being biosafe and biocompatible. ZnO thus has a wide range of applications in optoelectronics, intracellular nanosensors, transducers, energy conversion and medical sciences. This review relates specifically to intracellular electrochemical (glucose and free metal ion) biosensors based on functionalized zinc oxide nanowires/nanorods. For intracellular measurements, the ZnO nanowires/nanorods were grown on the tip of a borosilicate glass capillary (0.7 µm in diameter) and functionalized with membranes or enzymes to produce intracellular selective metal ion or glucose sensors. Successful intracellular measurements were carried out using ZnO nanowires/nanorods grown on small tips for glucose and free metal ions using two types of cells, human fat cells and frog oocytes. The sensors in this study were used to detect real-time changes of metal ions and glucose across human fat cells and frog cells using changes in the electrochemical potential at the interface of the intracellular micro-environment. Such devices are helpful in explaining various intracellular processes involving ions and glucose. PMID:26007730

  11. Assessment of Methods for the Intracellular Blockade of GABAA Receptors.

    PubMed

    Atherton, Laura A; Burnell, Erica S; Mellor, Jack R

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  12. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  13. On the Computing Potential of Intracellular Vesicles

    PubMed Central

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal ‘circuitry’ and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a ‘vesicle modification’ of the archetypal CBC ‘billiard ball model’ of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle ‘programming’ in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  14. Intracellular pH of symbiotic dinoflagellates

    NASA Astrophysics Data System (ADS)

    Gibbin, E. M.; Davy, S. K.

    2013-09-01

    Intracellular pH (pHi) is likely to play a key role in maintaining the functional success of cnidarian-dinoflagellate symbiosis, yet until now the pHi of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pHi over a daily light/dark cycle. The pHi of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHi 7.21-7.39 in the light and 7.06-7.14 in the dark. Of note, there was a significant genotypic difference in pHi, irrespective of irradiance.

  15. An intracellular anion channel critical for pigmentation

    PubMed Central

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. DOI: http://dx.doi.org/10.7554/eLife.04543.001 PMID:25513726

  16. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  17. Intracellular trafficking of heat shock factor 2.

    PubMed

    Le Goff, Pascale; Le Dréan, Yves; Le Péron, Christine; Le Jossic-Corcos, Catherine; Ainouche, Abdelkadder; Michel, Denis

    2004-04-01

    HSF2 is an enigmatic member of the heat shock factor family, identified in the homeotherm classes of birds and mammals. We report the characterization of HSF2 from an evolutionary ancient vertebrate, the fish rainbow trout (rtHSF2). rtHSF2 appears closely related to its mammalian counterparts at structural and functional levels. The conservation of the distinctive features of HSF2 from fish to human suggests that it should ensure important biological functions, not redundant with those of HSF1. Proteasome inhibition, reported as a potent stimulator of HSF2, leads to the stabilization and to a striking nuclear trafficking of rtHSF2-GFP fusion protein. Upon treatment with the proteasome inhibitor MG132, rtHSF2-GFP accumulates into PML nuclear bodies (NBs) independently of its sumoylation and, if expressed at moderate level, moves to nucleoli. The translocation of rtHSF2-GFP from NBs to nucleoli is greatly favored by overexpression of the heat shock protein Hsp70. The mammalian counterpart mouse HSF2 (mHSF2) also exhibited changes in intracellular distribution upon MG132 treatment. mHSF2 partitioned between a juxtanuclear area that we characterized as an aggresome and the nucleoli. These relocalizations are likely to reflect common structural changes of mouse and trout HSF2 upon activation. PMID:15023536

  18. Characterizations of intracellular arsenic in a bacterium

    NASA Astrophysics Data System (ADS)

    Wolfe-Simon, F.; Yannone, S. M.; Tainer, J. A.

    2011-12-01

    Life requires a key set of chemical elements to sustain growth. Yet, a growing body of literature suggests that microbes can alter their nutritional requirements based on the availability of these chemical elements. Under limiting conditions for one element microbes have been shown to utilize a variety of other elements to serve similar functions often (but not always) in similar molecular structures. Well-characterized elemental exchanges include manganese for iron, tungsten for molybdenum and sulfur for phosphorus or oxygen. These exchanges can be found in a wide variety of biomolecules ranging from protein to lipids and DNA. Recent evidence suggested that arsenic, as arsenate or As(V), was taken up and incorporated into the cellular material of the bacterium GFAJ-1. The evidence was interpreted to support As(V) acting in an analogous role to phosphate. We will therefore discuss our ongoing efforts to characterize intracellular arsenate and how it may partition among the cellular fractions of the microbial isolate GFAJ-1 when exposed to As(V) in the presence of various levels of phosphate. Under high As(V) conditions, cells express a dramatically different proteome than when grown given only phosphate. Ongoing studies on the diversity and potential role of proteins and metabolites produced in the presence of As(V) will be reported. These investigations promise to inform the role and additional metabolic potential for As in biology. Arsenic assimilation into biomolecules contributes to the expanding set of chemical elements utilized by microbes in unusual environmental niches.

  19. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  20. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. PMID:25513726

  1. A viral peptide for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

    2012-10-01

    Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

  2. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  3. Intracellular sphingosine releases calcium from lysosomes.

    PubMed

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. PMID:26613410

  4. Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

    PubMed Central

    Mendrola, Jeannine M.; Shi, Fumin; Park, Jin H.; Lemmon, Mark A.

    2013-01-01

    As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms. PMID:23863174

  5. Apoptotic cell death induced by intracellular proteolysis.

    PubMed

    Williams, M S; Henkart, P A

    1994-11-01

    To mimic the injection of granzymes into target cells by cytotoxic lymphocytes or the activation of endogenous proteases in programmed cell death, the proteases chymotrypsin, proteinase K, or trypsin were loaded into the cytoplasm of several different cell types using the osmotic lysis of pinosomes technique. Internalization of these proteases caused cell lysis within several hours, accompanied by extensive nuclear damage in most but not all combinations of target cells and proteases. This nuclear damage, quantitated by DNA release from nuclei, was associated with apoptotic features including DNA fragmentation into nucleosomal ladders, chromatin condensation, nuclear fragmentation, and membrane blebbing. Agents reported to block programmed cell death, including aurintricarboxylic acid, inhibitors of energy metabolism, and protein or RNA synthesis, failed to block this protease-induced death, although some inhibited nuclear damage. In separate experiments, introduction of staphylococcal nuclease into cells led to near complete (at least 75% of total) nucleosomal DNA fragmentation within 6 to 8 h. Condensation of chromatin did not accompany this fragmentation to the same extent, and there was approximately a 10-h lag between half-maximal DNA fragmentation and 50% loss of membrane integrity. The results suggest that activation of intracellular proteases during cell death by any molecular pathway could give rise to apoptotic morphology and DNA fragmentation. PMID:7930626

  6. On the Computing Potential of Intracellular Vesicles.

    PubMed

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  7. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    PubMed

    D'Amore, T; Panchal, C J; Stewart, G G

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures. PMID:3278685

  8. Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

    PubMed Central

    Duncan, R. Scott; Goad, Daryl L.; Grillo, Michael A.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. PMID:20335972

  9. Host metabolism regulates intracellular growth of Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Engel, Juan C; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A

    2013-01-16

    Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  10. Host metabolism regulates intracellular growth of Trypanosoma cruzi

    PubMed Central

    Caradonna, Kacey L.; Engel, Juan C.; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A.

    2012-01-01

    SUMMARY Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite’s replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  11. Intracellular Na+ kinetically interferes with the rotation of the Na(+)-driven flagellar motors of Vibrio alginolyticus.

    PubMed

    Yoshida, S; Sugiyama, S; Hojo, Y; Tokuda, H; Imae, Y

    1990-11-25

    To understand the mechanism of Na+ movement through the force-generating units of the Na(+)-driven flagellar motors of Vibrio alginolyticus, the effect of intracellular Na+ concentration on motor rotation was investigated. Control cells containing about 50 mM Na+ showed good motility even at 10 mM Na+ in the medium, i.e. in the absence of an inwardly directed Na+ gradient. In contrast, Na(+)-loaded cells containing about 400 mM Na+ showed very poor motility at 500 mM Na+ in the medium, i.e. even in the presence of an inwardly directed Na+ gradient. The membrane potential of the cells, which is a major driving force for the motor under these conditions, was not detectably altered, and consistently with this, Na(+)-coupled sucrose transport was only partly reduced in the Na(+)-loaded cells. Motility of the Na(+)-loaded cells was restored by decreasing the intracellular Na+ concentration, and the rate of restoration of motility correlated with the rate of the Na+ decrease. These results indicate that the absolute concentration of the intracellular Na+ is a determinant of the rotation rate of the Na(+)-driven flagellar motors of V. alginolyticus. A simple explanation for this phenomenon is that the force-generating unit of the motor has an intracellular Na(+)-binding site, at which the intracellular Na+ kinetically interferes with the rate of Na+ influx for motor rotation. PMID:2243095

  12. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  13. Global intracellular slow-wave dynamics of the thalamocortical system.

    PubMed

    Sheroziya, Maxim; Timofeev, Igor

    2014-06-25

    It is widely accepted that corticothalamic neurons recruit the thalamus in slow oscillation, but global slow-wave thalamocortical dynamics have never been experimentally shown. We analyzed intracellular activities of neurons either from different cortical areas or from a variety of specific and nonspecific thalamic nuclei in relation to the phase of global EEG signal in ketamine-xylazine anesthetized mice. We found that, on average, slow-wave active states started off within frontal cortical areas as well as higher-order and intralaminar thalamus (posterior and parafascicular nuclei) simultaneously. Then, the leading edge of active states propagated in the anteroposterior/lateral direction over the cortex at ∼40 mm/s. The latest structure we recorded within the slow-wave cycle was the anterior thalamus, which followed active states of the retrosplenial cortex. Active states from different cortical areas tended to terminate simultaneously. Sensory thalamic ventral posterior medial and lateral geniculate nuclei followed cortical active states with major inhibitory and weak tonic-like "modulator" EPSPs. In these nuclei, sharp-rising, large-amplitude EPSPs ("drivers") were not modulated by cortical slow waves, suggesting their origin in ascending pathways. The thalamic active states in other investigated nuclei were composed of depolarization: some revealing "driver"- and "modulator"-like EPSPs, others showing "modulator"-like EPSPs only. We conclude that sensory thalamic nuclei follow the propagating cortical waves, whereas neurons from higher-order thalamic nuclei display "hub dynamics" and thus may contribute to the generation of cortical slow waves. PMID:24966387

  14. Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

    PubMed

    Ivic, Lidija; Zhang, Cen; Zhang, Xinmin; Yoon, Sung Ok; Firestein, Stuart

    2002-01-01

    Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. PMID:11748633

  15. Insight into nanoparticle cellular uptake and intracellular targeting.

    PubMed

    Yameen, Basit; Choi, Won Il; Vilos, Cristian; Swami, Archana; Shi, Jinjun; Farokhzad, Omid C

    2014-09-28

    Collaborative efforts from the fields of biology, materials science, and engineering are leading to exciting progress in the development of nanomedicines. Since the targets of many therapeutic agents are localized in subcellular compartments, modulation of nanoparticle-cell interactions for efficient cellular uptake through the plasma membrane and the development of nanomedicines for precise delivery to subcellular compartments remain formidable challenges. Cellular internalization routes determine the post-internalization fate and intracellular localization of nanoparticles. This review highlights the cellular uptake routes most relevant to the field of non-targeted nanomedicine and presents an account of ligand-targeted nanoparticles for receptor-mediated cellular internalization as a strategy for modulating the cellular uptake of nanoparticles. Ligand-targeted nanoparticles have been the main impetus behind the progress of nanomedicines towards the clinic. This strategy has already resulted in remarkable progress towards effective oral delivery of nanomedicines that can overcome the intestinal epithelial barrier. A detailed overview of the recent developments in subcellular targeting as a novel platform for next-generation organelle-specific nanomedicines is also provided. Each section of the review includes prospects, potential, and concrete expectations from the field of targeted nanomedicines and strategies to meet those expectations. PMID:24984011

  16. Metabolic host responses to infection by intracellular bacterial pathogens

    PubMed Central

    Eisenreich, Wolfgang; Heesemann, Jürgen; Rudel, Thomas; Goebel, Werner

    2013-01-01

    The interaction of bacterial pathogens with mammalian hosts leads to a variety of physiological responses of the interacting partners aimed at an adaptation to the new situation. These responses include multiple metabolic changes in the affected host cells which are most obvious when the pathogen replicates within host cells as in case of intracellular bacterial pathogens. While the pathogen tries to deprive nutrients from the host cell, the host cell in return takes various metabolic countermeasures against the nutrient theft. During this conflicting interaction, the pathogen triggers metabolic host cell responses by means of common cell envelope components and specific virulence-associated factors. These host reactions generally promote replication of the pathogen. There is growing evidence that pathogen-specific factors may interfere in different ways with the complex regulatory network that controls the carbon and nitrogen metabolism of mammalian cells. The host cell defense answers include general metabolic reactions, like the generation of oxygen- and/or nitrogen-reactive species, and more specific measures aimed to prevent access to essential nutrients for the respective pathogen. Accurate results on metabolic host cell responses are often hampered by the use of cancer cell lines that already exhibit various de-regulated reactions in the primary carbon metabolism. Hence, there is an urgent need for cellular models that more closely reflect the in vivo infection conditions. The exact knowledge of the metabolic host cell responses may provide new interesting concepts for antibacterial therapies. PMID:23847769

  17. Insight into nanoparticle cellular uptake and intracellular targeting

    PubMed Central

    Yameen, Basit; Choi, Won Il; Vilos, Cristian; Swami, Archana; Shi, Jinjun; Farokhzad, Omid C.

    2014-01-01

    Collaborative efforts from the fields of biology, materials science, and engineering are leading to exciting progress in the development of nanomedicines. Since the targets of many therapeutic agents are localized in subcellular compartments, modulation of nanoparticle-cell interactions for an efficient cellular uptake through the plasma membrane, and the development of nanomedicines for precise delivery to subcellular compartments remain formidable challenges. The cellular internalization routes have a determining effect on the post-internalization fate and intracellular localization of nanoparticles. This review highlights the cellular uptake routes most relevant to the field of non-targeted nanomedicine, and presents an account of ligand targeted nanoparticles for receptor mediated cellular internalization as a strategy for modulating the cellular uptake of nanoparticles. Ligand targeted nanoparticles have been the main impetus behind the progress of nanomedicines towards the clinic. This strategy has even resulted in a remarkable development towards effective oral delivery of nanomedicines that can overcome the intestinal epithelial cellular barrier. A detailed overview of the recent developments towards subcellular targeting that is emerging as a platform for the next generation organelle specific nanomedicines is also provided. Each section of the review includes prospect, potential, and concrete expectations from the field of targeted nanomedicines and strategies to meet those expectations. PMID:24984011

  18. Intracellular magnetophoresis of amyloplasts and induction of root curvature

    NASA Technical Reports Server (NTRS)

    Kuznetsov, O. A.; Hasenstein, K. H.

    1996-01-01

    High-gradient magnetic fields (HGMFs) were used to induce intracellular magnetophoresis of amyloplasts. The HGMFs were generated by placing a small ferromagnetic wedge into a uniform magnetic field or at the gap edge between two permanent magnets. In the vicinity of the tip of the wedge the dynamic factor of the magnetic field, delta(H2/2), was about 10(9) Oe2.cm-1, which subjected the amyloplasts to a force comparable to that of gravity. When roots of 2-d-old seedlings of flax (Linum usitatissimum L.) were positioned vertically and exposed to an HGMF, curvature away from the wedge was transient and lasted approximately 1 h. Average curvature obtained after placing magnets, wedge and seedlings on a 1-rpm clinostat for 2 h was 33 +/- 5 degrees. Roots of horizontally placed control seedlings without rotation curved about 47 +/- 4 degrees. The time course of curvature and changes in growth rate were similar for gravicurvature and for root curvature induced by HGMFs. Microscopy showed displacement of amyloplasts in vitro and in vivo. Studies with Arabidopsis thaliana (L.) Heynh. showed that the wild type responded to HGMFs but the starchless mutant TC7 did not. The data indicate that a magnetic force can be used to study the gravisensing and response system of roots.

  19. Understanding anomalous delays in a model of intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Harvey, Emily; Kirk, Vivien; Osinga, Hinke M.; Sneyd, James; Wechselberger, Martin

    2010-12-01

    In many cell types, oscillations in the concentration of free intracellular calcium ions are used to control a variety of cellular functions. It has been suggested [J. Sneyd et al., "A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations," Proc. Natl. Acad. Sci. U.S.A. 103, 1675-1680 (2006)] that the mechanisms underlying the generation and control of such oscillations can be determined by means of a simple experiment, whereby a single exogenous pulse of inositol trisphosphate (IP3) is applied to the cell. However, more detailed mathematical investigations [M. Domijan et al., "Dynamical probing of the mechanisms underlying calcium oscillations," J. Nonlinear Sci. 16, 483-506 (2006)] have shown that this is not necessarily always true, and that the experimental data are more difficult to interpret than first thought. Here, we use geometric singular perturbation techniques to study the dynamics of models that make different assumptions about the mechanisms underlying the calcium oscillations. In particular, we show how recently developed canard theory for singularly perturbed systems with three or more slow variables [M. Wechselberger, "A propos de canards (Apropos canards)," Preprint, 2010] applies to these calcium models and how the presence of a curve of folded singularities and corresponding canards can result in anomalous delays in the response of these models to a pulse of IP3.

  20. Intracellular sodium homeostasis in rat hippocampal astrocytes.

    PubMed Central

    Rose, C R; Ransom, B R

    1996-01-01

    1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in

  1. Harmonization of the intracellular cytokine staining assay.

    PubMed

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables. PMID:22714399

  2. Use of photoproteins as intracellular calcium indicators.

    PubMed Central

    Blinks, J R

    1990-01-01

    The calcium-regulated photoproteins, of which aequorin is the best known, continue to be one of the most useful groups of intracellular Ca2+ indicators. They are self-contained bioluminescent systems that emit blue light in the presence of Ca2+ ions, can readily be purified intact, and are nontoxic when introduced into foreign cells. They have been used successfully as Ca2+ indicators in almost every kind of cell, but are most widely used in muscle cells because of their relative freedom from motion artifacts. Photoproteins have also been used in conjunction with microscopic image intensification to localize Ca2+ in cells. Their large molecular size makes them difficult to introduce into cells, but once there, they have the advantage of staying in the cytoplasm. Aequorin can be microinjected satisfactorily into single cells of almost any size, but a number of alternative methods for introducing photoproteins into cells have been developed in recent years. Disadvantages of the photoproteins for some applications include the nonlinear relation between [Ca2+] and light intensity, the modest speed with which they respond to sudden changes in [Ca2+], and the fact the Mg2+ antagonizes the effect of Ca2+. Native photoproteins consist of a mixture of isospecies, and there are differences in Ca2+ sensitivity and in kinetic properties--both among photoproteins and among the isospecies of a given photoprotein. The genes for several of the isospecies of aequorin have been cloned and expressed in E. coli. It seems reasonable to hope that genetic engineering techniques may soon make it possible to consider using, as Ca2+ indicators, rare isospecies or rare photoproteins that have optimal properties for particular applications. PMID:2190821

  3. Arrhythmogenic consequences of intracellular calcium waves.

    PubMed

    Xie, Lai-Hua; Weiss, James N

    2009-09-01

    Intracellular Ca(2+) (Ca(i)(2+)) waves are known to cause delayed afterdepolarizations (DADs), which have been associated with arrhythmias in cardiac disease states such as heart failure, catecholaminergic polymorphic ventricular tachycardia, and digitalis toxicity. Here we show that, in addition to DADs, Ca(i)(2+) waves also have other consequences relevant to arrhythmogenesis, including subcellular spatially discordant alternans (SDA, in which the amplitude of the local Ca(i)(2+) transient alternates out of phase in different regions of the same cell), sudden repolarization changes promoting the dispersion of refractoriness, and early afterdepolarizations (EADs). Ca(i)(2+) was imaged using a charge-coupled device-based system in fluo-4 AM-loaded isolated rabbit ventricular myocytes paced at constant or incrementally increasing rates, using either field stimulation, current clamp, or action potential (AP) clamp. Ca(i)(2+) waves were induced by Bay K 8644 (50 nM) + isoproterenol (100 nM), or low temperature. When pacing was initiated during a spontaneous Ca(i)(2+) wave, SDA occurred abruptly and persisted during pacing. Similarly, during rapid pacing, SDA typically arose suddenly from spatially concordant alternans, due to an abrupt phase reversal of the subcellular Ca(i)(2+) transient in a region of the myocyte. Ca(i)(2+) waves could be visualized interspersed with AP-triggered Ca(i)(2+) transients, producing a rich variety of subcellular Ca(i)(2+) transient patterns. With free-running APs, complex Ca(i)(2+) release patterns were associated with DADs, EADs, and sudden changes in AP duration. These findings link Ca(i)(2+) waves directly to a variety of arrhythmogenic phenomena relevant to the intact heart. PMID:19561309

  4. Targeting intracellular compartments by magnetic polymeric nanoparticles.

    PubMed

    Kocbek, Petra; Kralj, Slavko; Kreft, Mateja Erdani; Kristl, Julijana

    2013-09-27

    Superparamagnetic iron oxide nanoparticles (SPIONs) show a great promise for a wide specter of bioapplications, due to their characteristic magnetic properties exhibited only in the presence of magnetic field. Their advantages in the fields of magnetic drug targeting and imaging are well established and their safety is assumed, since iron oxide nanoparticles have already been approved for in vivo application, however, according to many literature reports the bare metal oxide nanoparticles may cause toxic effects on treated cells. Therefore, it is reasonable to prevent the direct interactions between metal oxide core and surrounding environment. In the current research ricinoleic acid coated maghemite nanoparticles were successfully synthesized, characterized and incorporated in the polymeric matrix, resulting in nanosized magnetic polymeric particles. The carrier system was shown to exhibit superparamagnetic properties and was therefore responsive towards external magnetic field. Bioevaluation using T47-D breast cancer cells confirmed internalization of magnetic polymeric nanoparticles (MNPs) and their intracellular localization in various subcellular compartments, depending on presence/absence of external magnetic field. However, the number of internalized MNPs observed by fluorescent and transmission electron microscopy was relatively low, making such way of targeting effective only for delivery of highly potent drugs. The scanning electron microscopy of treated cells revealed that MNPs influenced the cell adhesion, when external magnetic field was applied, and that treatment resulted in damaged apical plasma membrane right after exposure to the magnetic carrier. On the other hand, MNPs showed only reversibly reduced cellular metabolic activity in concentrations up to 200 μg/ml and, in the tested concentration the cell cycle distribution was within the normal range, indicating safety of the established magnetic carrier system for the treated cells. PMID:23603023

  5. ANOs 3–7 in the anoctamin/Tmem16 Cl− channel family are intracellular proteins

    PubMed Central

    Duran, Charity; Qu, Zhiqiang; Osunkoya, Adeboye O.; Cui, Yuanyuan

    2012-01-01

    Ca2+-activated Cl− channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3–7 in HEK293 cells did not generate Cl− currents activated by intracellular Ca2+, as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease. PMID:22075693

  6. Analysis of intracellular reducing levels in human hepatocytes on three-dimensional focusing microchip.

    PubMed

    Xu, Chunxiu; Cai, Longfei

    2014-02-01

    A novel three-dimensional hydrodynamic focusing microfluidic device integrated with high-throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6-tetramethyl-piperidine-1-oxyl-based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath-flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three-dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath-flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home-built laser-induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59-68 cells/min. PMID:23297173

  7. [Role of defective intracellular proteolysis in human degenerative diseases].

    PubMed

    Nezelof, Christian

    2012-11-01

    Although intracellular protein synthesis has been studied extensively, protein degradation and disposal, know as proteolysis, has been relatively neglected. Modern studies which led two Nobel prizes (de Duve in 1950 and Herschko, Rose and Ciechanover in 1980) established that proteolysis is ensured by two separate but complementary mechanisms: lysosomes responsible for auto and heterophagy and the Ubiquitin-Proteasome System (UPS). The UPS involves ubiquitin, a small molecule consisting of 76 amino acids found in all eukaryotic cells that ensures the identification of the protein to be degraded and its transport to the proteasome, an intracellular complex with enzymes which degrade unneeded or damaged proteins. The proteasome, acting as a composting agent, ensures the enzymatic dissociation of the protein. In this degradation process, as infinite screw, ubiquitin, peptides and amino acids are released and made available for a new cycle. Knowledge of the UPS and its related disorders is continually expanding. Concurrent with lysosomes which work in acidic environment, it is currently known that the UPS provides 80% to 90% of the proteolysis of the short-life proteins and ensures, as chaperon-molecules, the right conformation and hence the correct function of the proteins. The proteolytic activity generates abnormal residues (tau protein, amyloid and related proteins) and various soluble and insoluble wastes. Some are precipitated as inclusion-bodies or aggregosomes, identified years ago by pathologists. These aggregosomes affect almost exclusively long-lived cells (nervous and muscular, macophages). Pigment deposits, such as lipofuscines made by the peroxydation of cell membranes, are the most abundant. Due to their diverse chemical composition, they cannot be empoyed for a scientific classification. Failures of these systems are numerous. They vary not according to the chemical nature of the abnormal protein and wastes but the life span of the targeted cells and

  8. Antimicrobial susceptibility and molecular characterization of Mycobacterium intracellulare in China.

    PubMed

    Zhao, Xiuqin; Wang, Yufeng; Pang, Yu

    2014-10-01

    Mycobacterium avium complex (MAC) is the most common non-tuberculosis mycobacterial pathogen isolated from respiratory samples, mainly including two species, Mycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare). Although these two species belong to the same group, M. avium and M. intracellulare reveal significantly differences in pathogenicity and biology. Nevertheless, little is known regarding the drug resistant details profile of M. avium or M. intracellulare instead of MAC. Here, we examined the antimicrobial susceptibility profiles of 52 clinical M. intracellulare isolates against fourteen antimicrobial agents, which are widely selected for the treatment of nontuberculous mycobacteria (NTM) infection. The drug susceptibility test revealed that clarithromycin (47/52, 90.4%), rifampicin (41/52, 78.8%) and capreomycin (40/52, 76.9%) revealed highly antimicrobial activities against M. intracellulare isolates in vitro. Furthermore, all clarithromycin resistant isolates harbored mutations in the 23S rRNA gene, and the percentage of amikacin resistant ones with mutation in the rrs gene is 62.5% (10/16). The Hunter-Gaston Discriminatory Index (HGDI) value for the 16-loci Variable Number of Tandem Repeat (VNTR) typing of M. intracellulare isolates was 0.994, and M. intracellulare resistance to moxifloxacin was significantly more commonly found in clustered strains than in nonclustered strains (χ(2)=5.551, P=0.040). In conclusion, our data demonstrated that clarithromycin and capreomycin revealed highly antimicrobial activities against M. intracellulare isolates, and clarithromycin and amikacin resistance could be detected more readily and rapidly using molecular scanning of corresponding drug target than conventional drug susceptibility testing. We also found that infection by clustered strains was significantly associated with resistance to moxifloxacin. PMID:25131955

  9. Intracellular Acidosis Enhances the Excitability of Working Muscle

    NASA Astrophysics Data System (ADS)

    Pedersen, Thomas H.; Nielsen, Ole B.; Lamb, Graham D.; Stephenson, D. George

    2004-08-01

    Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

  10. Intracellular energetic units in red muscle cells.

    PubMed Central

    Saks, V A; Kaambre, T; Sikk, P; Eimre, M; Orlova, E; Paju, K; Piirsoo, A; Appaix, F; Kay, L; Regitz-Zagrosek, V; Fleck, E; Seppet, E

    2001-01-01

    The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic

  11. Vesicular demyelination induced by raised intracellular calcium.

    PubMed

    Smith, K J; Hall, S M; Schauf, C L

    1985-11-01

    myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca2+ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced. PMID:3003255

  12. Intracellular markers in acute myeloid leukemia diagnosis.

    PubMed

    Koníková, E; Glasová, M; Kusenda, J; Babusíková, O

    1998-01-01

    In our study we used a new proposed system of CD45 monoclonal antibody in combination with the side scatter (SSC) parameter as a very useful gating method allowing myeloblast detection especially in cases with low blasts percentage in examined samples. Immunological demonstration of myeloperoxidase (MPO) in the cytoplasm of AML blasts is considered to be a reliable and highly sensitive marker. Using a direct single and double immunofluorescence staining method and flow cytometry we evaluated the intracellular expression of two granular constituents of myeloid cells--MPO and lactoferrin (LF) in leukemia cells from 18 patients at AML diagnosis, two patients in remission after allogenic bone marrow transplantation and in six controls. Two different fixation/permeabilization techniques were used: Fix&Perm, paraformaldehyde and saponin prior to monoclonal antibody staining in order to verify the sensitivity of two labeling methods for MPO. Although both reagents used in this study proved to be efficient tools for the fixation and permeabilization of leukemia cells, the second one was characterized by higher sensitivity in detection of MPO. By double staining of MPO and LF we were able to distinguish undifferentiated cells from the granulomonocytic maturation compartments in bone marrow, since LF is proposed to be selectively expressed from the myelocyte stage of differentiation onward. Cytoplasmic CD13 expression was detectable in AML blasts after their buffered-formaldehyde-acetone fixation/permeabilization. According to our results the detection of MPO and CD13 markers in the cytoplasm of leukemia cells is of great importance in the definition of FAB M0-M1 subtype of AML. Furthermore we described overexpression of CD34 antigen in AML and revealed the characteristic marker combination when CD34 was studied simultaneously with MPO. This finding also coincided with some atypical phenotypic features (CD15/MPO, CD7/cCD13, CD2/cCD13, CD33/cCD13, MPO/cCD13) contributing to

  13. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  14. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

    PubMed Central

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R.; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Do Heo, Won; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  15. Cellular Metabolic Activity and the Oxygen and Hydrogen Stable Isotope Composition of Intracellular Water and Metabolites

    NASA Astrophysics Data System (ADS)

    Kreuzer-Martin, H. W.; Hegg, E. L.

    2008-12-01

    Intracellular water is an important pool of oxygen and hydrogen atoms for biosynthesis. Intracellular water is usually assumed to be isotopically identical to extracellular water, but an unexpected experimental result caused us to question this assumption. Heme O isolated from Escherichia coli cells grown in 95% H218O contained only a fraction of the theoretical value of labeled oxygen at a position where the O atom was known to be derived from water. In fact, fewer than half of the oxygen atoms were labeled. In an effort to explain this surprising result, we developed a method to determine the isotope ratios of intracellular water in cultured cells. The results of our experiments showed that during active growth, up to 70% of the oxygen atoms and 50% of the hydrogen atoms in the intracellular water of E. coli are generated during metabolism and can be isotopically distinct from extracellular water. The fraction of isotopically distinct atoms was substantially less in stationary phase and chilled cells, consistent with our hypothesis that less metabolically-generated water would be present in cells with lower metabolic activity. Our results were consistent with and explained the result of the heme O labeling experiment. Only about 40% of the O atoms on the heme O molecule were labeled because, presumably, only about 40% of the water inside the cells was 18O water that had diffused in from the culture medium. The rest of the intracellular water contained 16O atoms derived from either nutrients or atmospheric oxygen. To test whether we could also detect metabolically-derived hydrogen atoms in cellular constituents, we isolated fatty acids from log-phase and stationary phase E. coli and determined the H isotope ratios of individual fatty acids. The results of these experiments showed that environmental water contributed more H atoms to fatty acids isolated in stationary phase than to the same fatty acids isolated from log-phase cells. Stable isotope analyses of

  16. The interferon response to intracellular DNA: why so many receptors?

    PubMed

    Unterholzner, Leonie

    2013-11-01

    The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. PMID:23962476

  17. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungalrelated parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and...

  18. Intracellular calcium ions as regulators of renal tubular sodium transport.

    PubMed

    Windhager, E; Frindt, G; Yang, J M; Lee, C O

    1986-09-15

    This review addresses the putative role of intracellular calcium ions in the regulation of sodium transport by renal tubules. Cytoplasmic calcium-ion activities in proximal tubules of Necturus are less than 10(-7) M and can be increased by lowering the electrochemical potential gradient for sodium ions across the peritubular cell membrane, or by addition of quinidine or ionomycin to peritubular fluid. Whereas lowering of the peritubular Na concentration increases cytosolic [Ca++] and [H+], ionomycin, a calcium ionophore, raises intracellular [Ca++] without decreasing pHi. The intracellular calcium-ion level is maintained by transport processes in the plasma membrane and membranes of intracellular organelles, as well as by calcium-binding proteins. Calcium ions inhibit net transport of sodium by reducing the rate of sodium entry across the luminal cell membrane. In the collecting tubule this inhibition is caused, at least in part, by an indirect reduction in the activity of the amiloride-sensitive sodium channel. PMID:2430134

  19. EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

    EPA Science Inventory

    Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

  20. Stress-induced inhibition of nonsense mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11

    PubMed Central

    Martin, Leenus; Gardner, Lawrence B.

    2014-01-01

    SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response. PMID:25399695

  1. Assessment of Methods for the Intracellular Blockade of GABAA Receptors

    PubMed Central

    Atherton, Laura A.; Burnell, Erica S.; Mellor, Jack R.

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4’-dinitro-stilbene-2,2’-disulphonic acid (DNDS) and 4,4’-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  2. Intracellular Renin Disrupts Chemical Communication between Heart Cells. Pathophysiological Implications

    PubMed Central

    De Mello, Walmor C.

    2015-01-01

    Highlights Intracellular renin disrupts chemical communication in the heartAngiotensinogen enhances the effect of reninIntracellular enalaprilat reduces significantly the effect of reninIntracellular renin increases the inward calcium currentHarmful versus beneficial effect during myocardial infarction The influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; (1) under normal conditions, Lucifer Yellow flows from cell to cell through gap junctions; (2) the intracellular dialysis of renin (100 nM) disrupts chemical communication – an effect enhanced by simultaneous administration of angiotensinogen (100 nM); (3) enalaprilat (10−9 M) administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; (4) aliskiren (10−8 M) inhibited the effect of renin on chemical communication; (5) the possible role of intracellular renin independently of angiotensin II (Ang II) was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; (6) the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed; (7) the present results indicate that intracellular renin due to internalization or in situ synthesis causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function. PMID:25657639

  3. [Intracellular and extracellular functions of phosphorus compound in the body].

    PubMed

    Segawa, Hiroko; Hanazaki, Ai; Miyamoto, Ken-ichi

    2016-02-01

    Phosphorus, as a phosphate is a component of bone, cellular membrane, and also high-energy phosphate compounds, and nucleic acids. Also phosphate acts as a buffer to maintain the pH and is concerned with functional regulation of several proteins and intracellular signaling through the phosphorylation/dephosphorylation. Thus phosphorus plays a variety of important roles intracellular and extracellular component. A disorder of phosphate homeostasis results bone disorder and general metabolic dysfunction of all body tissues and organs. PMID:26813497

  4. Intracellular calcium store filling by an L-type calcium current in the basolateral amygdala at subthreshold membrane potentials

    PubMed Central

    Power, John M; Sah, Pankaj

    2005-01-01

    The long-term changes that underlie learning and memory are activated by rises in intracellular Ca2+ that activate a number of signalling pathways and trigger changes in gene transcription. Ca2+ rises due to influx via L-type voltage-dependent Ca2+ channels (L-VDCCs) and release from intracellular Ca2+ stores have been consistently implicated in the biochemical cascades that underlie the final changes in memory formation. Here, we show that pyramidal neurones in the basolateral amygdala express an L-VDCC that is active at resting membrane potentials. Subthreshold depolarization of neurones either by current injection or summating synaptic potentials led to a sustained rise in cytosolic Ca2+ that was blocked by the dihydropyridine nicardipine. Activation of metabotropic receptors released Ca2+ from intracellular Ca2+ stores. At hyperpolarized potentials, metabotropic-evoked store release ran down with repeated stimulation. Depolarization of cells to −50 mV, or maintaining them at the resting membrane potential, restored release from intracellular Ca2+ stores, an effect that was blocked by nicardipine. These results show that Ca2+ influx via a low-voltage-activated L-type Ca2+ current refills inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores, and maintains Ca2+ release and wave generation by metabotropic receptor activation. PMID:15550460

  5. Proton-dependent zinc release from intracellular ligands

    PubMed Central

    Kiedrowski, Lech

    2014-01-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn2+ into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn2+. Although exposure to the protonophore FCCP precludes re-loading of the mitochondria and acidic organelles with Zn2+, FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn2+. Therefore, Zn2+-releasing stores were not mitochondria or acidic organelles but rather intracellular Zn2+ ligands. To test which ligands might be involved, the rate of acid-induced Zn2+ release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn2+] was monitored in vitro using the ratiometric Zn2+-sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn2+ but did chelate Cu2+; the remaining ligands chelated Zn2+ and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn2+ release. The zinc-cysteine complexes also released Zn2+ when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn2+, these complexes may represent the stores responsible for an acid-induced intracellular Zn2+ release. PMID:24606401

  6. Invasion of the Central Nervous System by Intracellular Bacteria

    PubMed Central

    Drevets, Douglas A.; Leenen, Pieter J. M.; Greenfield, Ronald A.

    2004-01-01

    Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens. PMID:15084504

  7. Molecular targeting of intracellular compartments specifically in cancer cells.

    PubMed

    Pandya, Hetal; Gibo, Denise M; Debinski, Waldemar

    2010-05-01

    We have implemented a strategy in which a genetically engineered, single-chain protein specifically recognizes cancer cells and is trafficked to a targeted subcellular compartment, such as the nucleus. The recombinant protein termed IL-13.E13K-D2-NLS has a triple functional property: (1) it binds a cancer-associated receptor, interleukin 13 receptor alpha 2 (IL-13Rα2), using modified IL-13 ligand, IL-13.E13K; (2) it exports its C-terminal portion out of the endosomal compartment using Pseudomonas aeruginosa exotoxin A (PE) translocation domain (D2); and (3) it travels to and accumulates in the nucleus guided by the nuclear localization signal (NLS). Here, we have demonstrated that this protein is transported into the brain tumor cells' nucleus, using 3 different methods of protein conjugation to dyes for the purpose of direct visualization of the protein's intracellular trafficking. IL-13.E13K-D2-NLS, and not the controls such as IL-13.E13K-D2, IL-13.E13K-NLS, or IL-13.E13K, accumulated in nuclei very efficiently, which increased with the time the cells were exposed to the protein. Also, IL-13.E13K-D2-NLS did not exhibit nuclear transport in cells with low expression levels of IL-13Rα2. Thus, it is possible to recognize cancer cells through their specific receptors and deliver a conjugated protein that travels specifically to the nucleus. Hence, our molecular targeting strategy succeeded in generating a single-chain proteinaceous agent capable of delivering drugs/labels needed to be localized to the cells' nuclei or potentially any other subcellular compartment, for their optimal efficacy or ability to exert their specific action. PMID:20740056

  8. Molecular Targeting of Intracellular Compartments Specifically in Cancer Cells

    PubMed Central

    Pandya, Hetal; Gibo, Denise M.; Debinski, Waldemar

    2010-01-01

    We have implemented a strategy in which a genetically engineered, single-chain protein specifically recognizes cancer cells and is trafficked to a targeted subcellular compartment, such as the nucleus. The recombinant protein termed IL-13.E13K-D2-NLS has a triple functional property: (1) it binds a cancer-associated receptor, interleukin 13 receptor alpha 2 (IL-13Rα2), using modified IL-13 ligand, IL-13.E13K; (2) it exports its C-terminal portion out of the endosomal compartment using Pseudomonas aeruginosa exotoxin A (PE) translocation domain (D2); and (3) it travels to and accumulates in the nucleus guided by the nuclear localization signal (NLS). Here, we have demonstrated that this protein is transported into the brain tumor cells’ nucleus, using 3 different methods of protein conjugation to dyes for the purpose of direct visualization of the protein’s intracellular trafficking. IL-13.E13K-D2-NLS, and not the controls such as IL-13.E13K-D2, IL-13.E13K-NLS, or IL-13.E13K, accumulated in nuclei very efficiently, which increased with the time the cells were exposed to the protein. Also, IL-13.E13K-D2-NLS did not exhibit nuclear transport in cells with low expression levels of IL-13Rα2. Thus, it is possible to recognize cancer cells through their specific receptors and deliver a conjugated protein that travels specifically to the nucleus. Hence, our molecular targeting strategy succeeded in generating a single-chain proteinaceous agent capable of delivering drugs/labels needed to be localized to the cells’ nuclei or potentially any other subcellular compartment, for their optimal efficacy or ability to exert their specific action. PMID:20740056

  9. Intracellular fate of spherical nucleic acid nanoparticle conjugates.

    PubMed

    Wu, Xiaochen A; Choi, Chung Hang J; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A

    2014-05-28

    Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome

  10. Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils.

    PubMed

    Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Bolonheis, Simone Marques; Ferreira, Zulma F; Muscará, Marcelo Nicolas; Teixeira, Simone Aparecida; Farsky, Sandra Helena Poliselli

    2012-11-01

    Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 μM; 2 h) and subsequently incubated with LPS (5 μg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or β(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure. PMID:22717997

  11. Cyst formation following disruption of intracellular calcium signaling

    PubMed Central

    Kuo, Ivana Y.; DesRochers, Teresa M.; Kimmerling, Erica P.; Nguyen, Lily; Ehrlich, Barbara E.; Kaplan, David L.

    2014-01-01

    Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca2+) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca2+-activated Ca2+ channel of the endoplasmic reticulum. We hypothesize that Ca2+ signaling through PC2, or other intracellular Ca2+ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca2+ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca2+ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca2+ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca2+ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca2+ signaling lead to ADPKD cysts. PMID:25228769

  12. Dysregulation of intracellular trafficking and endosomal sorting in Alzheimer's disease: controversies and unanswered questions.

    PubMed

    Toh, Wei Hong; Gleeson, Paul A

    2016-07-15

    Alzheimer's disease (AD) is characterized by the accumulation of amyloid plaques in the brain consisting of an aggregated form of amyloid β-peptide (Aβ) derived from sequential amyloidogenic processing of the amyloid precursor protein (APP) by membrane-bound proteases β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase. The initial processing of APP by BACE1 is re-gulated by intracellular sorting events of the enzyme, which is a prime target for therapeutic intervention. GWAS (genome-wide sequencing studies) have identified several AD-susceptibility genes that are associated with the regulation of membrane trafficking, and substantial evidence now indicates that AD is likely to arise from defective membrane trafficking in either or both of the secretory and endocytic pathways. Considerable progress has been made in defining the intracellular trafficking pathways of BACE1 and APP and the sorting signals of these membrane proteins that define their itineraries. In this review we highlight recent advances in understanding the regulation of the intracellular sorting of BACE1 and APP, discuss how dysregulation of these trafficking events may lead to enhanced generation of the neurotoxic Aβ products in AD and highlight the unresolved questions in the field. PMID:27407168

  13. Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance.

    PubMed

    Berti, Denise A; Russo, Lilian C; Castro, Leandro M; Cruz, Lilian; Gozzo, Fábio C; Heimann, Joel C; Lima, Fabio B; Oliveira, Ariclécio C; Andreotti, Sandra; Prada, Patrícia O; Heimann, Andrea S; Ferro, Emer S

    2012-08-01

    Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high-caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high-caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin-stimulated glucose uptake in 3T3-L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin-induced glucose uptake in 3T3-L1 adipocytes. PMID:22740317

  14. Intracellular oxidative stress and cytotoxicity in rat primary cortical neurons exposed to cholesterol secoaldehyde.

    PubMed

    Raghavamenon, Achuthan C; Gernapudi, Ramkishore; Babu, Sainath; D'Auvergne, Oswald; Murthy, Subramanyam N; Kadowitz, Philip J; Uppu, Rao M

    2009-08-14

    Cholesterol secoaldehyde (ChSeco or 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al) has been shown to induce Abeta aggregation and apoptosis in GT1-7 hypothalamic neurons. The present study was undertaken to evaluate the effects of ChSeco on rat primary cortical neuronal cells. ChSeco was cytotoxic at concentrations ranging from 5 to 20 microM, while cholesterol of comparable concentrations showed little or no toxicity. In ChSeco-exposed neuronal cells, there was an increased formation of intracellular peroxide or peroxide-like substance(s), the levels of which were comparable to those found in typical menadione exposures. There was a loss in the mitochondrial transmembrane potential, the extent of which was dependent on concentration of ChSeco employed. Pre-treatment with N-acetyl-L-cysteine (5 mM; 1 h) offered protection against the cytotoxicity and the generation of intracellular oxidants. Cytotoxicity of ChSeco was evidenced by the loss of axonal branches and also condensed apoptotic nuclei in these cells. Immunohistochemical analysis revealed a decreased intracellular Abeta42 staining proportional to the loss in the axonal out growth and dendritic branches. The observed decrease in Abeta42 has been suggested to be due to loss of integrity of dendrites and the plasma membrane, possibly resulting from increased production of reactive oxygen species. PMID:19505436

  15. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

    PubMed Central

    Zamaleeva, Alsu I.; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne

    2015-01-01

    Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging. PMID:26404317

  16. Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

    PubMed

    Ottonello, L; Gonella, R; Dapino, P; Sacchetti, C; Dallegri, F

    1998-08-01

    Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis. PMID:9694511

  17. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles.

    PubMed

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  18. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology. PMID:23826393

  19. EBP50 induces apoptosis in macrophages by upregulating nitric oxide production to eliminate intracellular Mycobacterium tuberculosis

    PubMed Central

    Guo, Yang; Deng, Yating; Huang, Zikun; Luo, Qing; Peng, Yiping; Chen, Jie; Jiang, Hong; Ye, Jianqing; Li, Junming

    2016-01-01

    Mycobacterium bovis BCG is known to have the capacity to inhibit the positioning of iNOS on BCG-containing phagosomes by interfering with EBP50, a scaffolding protein that controls the recruitment of inducible nitric oxide synthase (iNOS) at the vicinity of phagosomes in macrophages. However, knockdown of the expression of EBP50 still facilitates the intracellular survival of BCG, which suggested that EBP50 may have some other unknown antimycobacterial properties. In this study we show that overexpression of EBP50 by a recombinant lentivirus had no effect on the iNOS recruitment to M.tuberculosis-containing phagosomes, but significantly promoted the elimination of intracellular M.tuberculosis. We revealed in the present study that the enhancement of intracellular killing to M. tuberculosis upon EBP50 overexpression was due to the increased level of apoptosis in macrophages. We showed that EBP50 overexpression significantly increased the expression of iNOS and generation of nitric oxide (NO), and EBP50-induced apoptosis was NO-dependent and mediated by Bax and caspase-3. We found that M. tuberculosis decreases while Mycobacterium smegmatis increases the expression of EBP50 in RAW264.7 cells, which suggested that virulent mycobacteria are capable of modulating the antimycobacterial properties of macrophages by inhibiting the expression and interfering with the function of EBP50. PMID:26729618

  20. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    PubMed Central

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  1. Overview on how oncogenic Kras promotes pancreatic carcinogenesis by inducing low intracellular ROS levels.

    PubMed

    Kong, Bo; Qia, Chengjia; Erkan, Mert; Kleeff, Jörg; Michalski, Christoph W

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease without clearly known disease causes. Recent epidemiological and animal studies suggest that the supplementation of dietary antioxidants (e.g., vitamins C and E) decreases cancer risk, implying that increased reactive oxygen species (ROS) may play a role in pancreatic carcinogenesis. However, oncogenic Kras mutations (e.g., Kras(G12D)), which are present in more than 90% of PDAC, have been proven to foster low intracellular ROS levels. Here, oncogenic Kras activates expression of a series of anti-oxidant genes via Nrf2 (nuclear factor, erythroid derived 2, like 2) and also mediates an unusual metabolic pathway of glutamine to generate NADPH. This can then be used as the reducing power for ROS detoxification, leading collectively to low ROS levels in pancreatic pre-neoplastic cells and in cancer cells. In adult stem cells and cancer stem cells, low ROS levels have been associated with the formation of a proliferation-permissive intracellular environment and with perseverance of self-renewal capacities. Therefore, it is conceivable that low intracellular ROS levels may contribute significantly to oncogenic Kras-mediated PDAC formation. PMID:24062691

  2. Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris.

    PubMed

    Yin, Huixiang; Liu, Zhenwang; Zhang, Ailian; Zhang, Tianyuan; Luo, Jinxian; Shen, Jincheng; Chen, Liping; Zhou, Bing; Fu, Xian; Fu, Ceyi; Zhang, Zehua

    2012-08-01

    Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700 μg/ml). D-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50 L bioreactor at a 20 L working volume. After 48 h fermentation with continuous feeding of 25% (w/v) D-sorbitol and 0.8% PTM4, the cell grew to A(600)=178 and intracellularly expressed Canstatin-N reached 780 mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340). PMID:22575729

  3. Identification of phenolics in litchi and evaluation of anticancer cell proliferation activity and intracellular antioxidant activity.

    PubMed

    Wen, Lingrong; You, Lijun; Yang, Xiaoman; Yang, Jiali; Chen, Feng; Jiang, Yueming; Yang, Bao

    2015-07-01

    Litchi leaf is a good resource for phenolics, which are good candidates for medicines. In this work, three phenolics were isolated from litchi leaf by column chromatography. Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy as secoisolariciresinol 9'-O-β-D-xyloside (1), 4,7,7',8',9,9'-hexahydroxy-3,3'-dimethoxy-8,4'-oxyneolignan (2), and cinnamtannin B1 (3). Cinnamtannin B1 showed better extra- and intracellular antioxidant activities than Compounds 1 and 2. The intracellular antioxidant activity of cinnamtannin B1 was related to the upregulation of endogenous antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase), and inhibition of ROS generation. Furthermore, cinnamtannin B1 exhibited strong antiproliferative effects against HepG2 and Siha cell lines with no significant cytotoxicities. In the case of the HepG2 cell line, cell cycle arrest and apoptosis induction were the underlying anticancer mechanisms of cinnamtannin B1. The results indicated that cinnamtannin B1 was a potent cancer cell proliferation inhibitor and a good intracellular antioxidant. PMID:25857215

  4. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

    PubMed Central

    Abraham, Ambily; Natraj, Usha; Karande, Anjali A.; Gulati, Ashutosh; Murthy, Mathur R. N.; Murugesan, Sathyabalan; Mukunda, Pavithra; Savithri, Handanahal S.

    2016-01-01

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies–D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies. PMID:26905902

  5. Control, exploitation and tolerance of intracellular noise.

    SciTech Connect

    Rao, Chris V.; Wolf, Denise M.; Arkin, Adam P.

    2002-11-14

    Noise has many roles in biological function, including generation of errors in DNA replication leading to mutation and evolution, noise-driven divergence of cell fates, noise-induced amplification of signals, and maintenance of the quantitative individuality of cells. Yet there is order to the behavior and development of cells. They operate within strict parameters and in many cases this behavior seems robust, implying that noise is largely filtered by the system. How can we explain the use, rejection and sensitivity to noise that is found in biological systems? An exploration of the sources and consequences of noise calls for the use of stochastic models.

  6. Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Voth, Daniel E.; Howe, Dale; Cockrell, Diane C.; Bastidas, Robert J.; Valdivia, Raphael H.

    2014-01-01

    The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion. We predict additional C. burnetii effectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termed Coxiella vacuolar protein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins. C. burnetii ΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpE mutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpD and ΔcvpE mutants rescued intracellular growth and PV generation, whereas the growth of C. burnetii ΔcvpB and ΔcvpC was rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicate C. burnetii encodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages. PMID:25422265

  7. Early Events in Phagosome Establishment Are Required for Intracellular Survival of Legionella pneumophila

    PubMed Central

    Wiater, Lawrence A.; Dunn, Kenneth; Maxfield, Frederick R.; Shuman, Howard A.

    1998-01-01

    During infection, the Legionnaires’ disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism’s ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication. PMID:9712800

  8. Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro

    PubMed Central

    Canfrán-Duque, Alberto; Pastor, Oscar; Reina, Manuel; Lerma, Milagros; Cruz-Jentoft, Alfonso J.

    2015-01-01

    Scope First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation. Methods HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation. Results Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics. Conclusion Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials. PMID:26517556

  9. Ethambutol plasma and intracellular pharmacokinetics: A pharmacogenetic study.

    PubMed

    Fatiguso, Giovanna; Allegra, Sarah; Calcagno, Andrea; Baietto, Lorena; Motta, Ilaria; Favata, Fabio; Cusato, Jessica; Bonora, Stefano; Perri, Giovanni Di; D'Avolio, Antonio

    2016-01-30

    We evaluated ethambutol plasma and intracellular pharmacokinetic according to single nucleotide polymorphisms in ABCB1, OATP1B1, PXR, VDR, CYP24A1 and CYP27B1 genes. Mycobacterium tubercolosis infected patients were enrolled. Standard weight-adjusted antitubercular treatment was administered intravenously for 2 weeks and then orally. Allelic discrimination was performed by real-time PCR. Ethambutol plasma and intracellular concentrations were measured by UPLC-MS/MS methods. Twenty-four patients were included. Considering weeks 2 and 4, median plasma Ctrough were 73 ng/mL and 247 ng/mL, intracellular Ctrough were 16,863 ng/mL and 13,535 ng/mL, plasma Cmax were 5627 ng/mL and 2229 ng/mL, intracellular Cmax were 133,830 ng/mL and 78,544 ng/mL. At week 2, ABCB1 3435 CT/TT (p=0.023) and CYP24A1 8620 AG/GG (p=0.030) genotypes for plasma Ctrough, BsmI AA (p=0.036) for intracellular Ctrough and BsmI AA (p<0.001) and ApaI AA (p=0.048) for intracellular Cmax, remained in linear regression analysis as predictive factors. Concerning week 4 only ABCB1 3435 CT/TT (p=0.035) and Cdx2 AG/GG (p=0.004) genotypes for plasma Ctrough and BsmI AA (p=0.028) for plasma Cmax were retained in final regression model. We reveal, for the first time, the possible role of single nucleotide polymorphisms on ethambutol plasma and intracellular concentrations; this may further the potential use of pharmacogenetic for tailoring antitubercular treatment. PMID:26642947

  10. An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

    PubMed Central

    Borg, Sarah; Popp, Felix; Hofmann, Julia; Leonhardt, Heinrich; Rothbauer, Ulrich

    2015-01-01

    ABSTRACT  Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. Importance   Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for

  11. Strategies for the intracellular delivery of nanoparticles.

    PubMed

    Chou, Leo Y T; Ming, Kevin; Chan, Warren C W

    2011-01-01

    The ability to target contrast agents and therapeutics inside cells is becoming important as we strive to decipher the complex network of events that occur within living cells and design therapies that can modulate these processes. Nanotechnology researchers have generated a growing list of nanoparticles designed for such applications. These particles can be assembled from a variety of materials into desirable geometries and configurations and possess useful properties and functionalities. Undoubtedly, the effective delivery of these nanomaterials into cells will be critical to their applications. In this tutorial review, we discuss the fundamental challenges of delivering nanoparticles into cells and to the targeted organelles, and summarize strategies that have been developed to-date. PMID:20886124

  12. Excitation and Inhibition Compete to Control Spiking during Hippocampal Ripples: Intracellular Study in Behaving Mice

    PubMed Central

    English, Daniel F.; Peyrache, Adrien; Stark, Eran; Roux, Lisa; Vallentin, Daniela; Long, Michael A.

    2014-01-01

    High-frequency ripple oscillations, observed most prominently in the hippocampal CA1 pyramidal layer, are associated with memory consolidation. The cellular and network mechanisms underlying the generation of the rhythm and the recruitment of spikes from pyramidal neurons are still poorly understood. Using intracellular, sharp electrode recordings in freely moving, drug-free mice, we observed consistent large depolarizations in CA1 pyramidal cells during sharp wave ripples, which are associated with ripple frequency fluctuation of the membrane potential (“intracellular ripple”). Despite consistent depolarization, often exceeding pre-ripple spike threshold values, current pulse-induced spikes were strongly suppressed, indicating that spiking was under the control of concurrent shunting inhibition. Ripple events were followed by a prominent afterhyperpolarization and spike suppression. Action potentials during and outside ripples were orthodromic, arguing against ectopic spike generation, which has been postulated by computational models of ripple generation. These findings indicate that dendritic excitation of pyramidal neurons during ripples is countered by shunting of the membrane and postripple silence is mediated by hyperpolarizing inhibition. PMID:25471587

  13. Proton-dependent zinc release from intracellular ligands.

    PubMed

    Kiedrowski, Lech

    2014-07-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn(2+) into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn(2+) . Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn(2+) , FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn(2+) . Therefore, Zn(2+) -releasing stores were not mitochondria or acidic organelles but rather intracellular Zn(2+) ligands. To test which ligands might be involved, the rate of acid-induced Zn(2+) release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn(2+) ] was monitored in vitro using the ratiometric Zn(2+) -sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn(2+) but did chelate Cu(2+) ; the remaining ligands chelated Zn(2+) and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn(2+) release. The zinc-cysteine complexes also released Zn(2+) when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn(2+) , these complexes may represent the stores responsible for an acid-induced intracellular Zn(2+) release. This study aimed at identifying intracellular stores which release Zn(2+) when pHi drops from 6.6 to 6.1. It was found that these stores are not mitochondria or acidic organelles, but rather intracellular Zn(2+) ligands. When the pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes showed a rapid acceleration in the rate of Zn(2+) release. Therefore, the stores responsible for an acid-induced intracellular Zn(2+) release in neurons may be the cytosolic zinc-cysteine complexes

  14. Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender.

    PubMed

    Singh, V K; Atreja, S K; Kumar, R; Chhillar, S; Singh, A K

    2012-08-01

    In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa. PMID:21988572

  15. Intracellular delivery of molecules using microfabricated nanoneedle arrays.

    PubMed

    Park, Seonhee; Choi, Seong-O; Paik, Seung-joon; Choi, Seungkeun; Allen, Mark; Prausnitz, Mark

    2016-02-01

    Many bioactive molecules have intracellular targets, but have difficulty crossing the cell membrane to reach those targets. To address this difficulty, we fabricated arrays of nanoneedles to gently and simultaneously puncture 10(5) cells and thereby provide transient pathways for transport of molecules into the cells. The nanoneedles were microfabricated by etching silicon to create arrays of nanoneedles measuring 12 μm in height, tapering to a sharp tip less than 30 nm wide to facilitate puncture into cells and spaced 10 μm apart in order to have at least one nanoneedle puncture each cell in a confluent monolayer. These nanoneedles were used for intracellular delivery in two ways: puncture loading, in which nanoneedle arrays were pressed into cell monolayers, and centrifuge loading, in which cells in suspension were spun down onto nanoneedle arrays. The effects on intracellular uptake and cell viability were determined as a function of nanoneedle length and sharpness, puncture force and duration, and molecular weight of the molecule delivered. Under optimal conditions, intracellular uptake was seen in approximately 50 % of cells while maintaining high cell viability. Overall, this study provides a comparative analysis of intracellular delivery using nanoneedle arrays by two different loading methods over a range of operating parameters. PMID:26797026

  16. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

    PubMed

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-07-29

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria. PMID:25009182

  17. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  18. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  19. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila.

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  20. Intracellular delivery of and sensing with quantum dot bioconjugates

    NASA Astrophysics Data System (ADS)

    Delehanty, James B.; Bradburne, Christopher E.; Medintz, Igor L.; Farrell, Dorothy; Pons, Thomas; Deschamps, Jeffrey R.; Brunel, Florence M.; Dawson, Philip E.; Mattoussi, Hedi

    2009-02-01

    Luminescent semiconductor quantum dots (QDs) possess several unique optical properties that suggest they will be superior reagents compared to traditional organic fluorophores for applications such as the labeling of subcellular structures and the sensing of biological processes within living cells. Chief among these properties are their 1) high quantum yields, 2) broad absorption spectra coupled with narrow symmetric, size-tunable emissions and 3) the ability to excite multiple QD populations at a single wavelength removed from emission. This latter attribute presents the exciting possibility of multiplexed or "multicolor" intracellular imaging and sensing. In order for QDs to reach their full potential as intracellular imaging and sensing reagents, however, facile and robust methodologies for delivering QDs in a controlled manner to specific subcellular locations must be developed. We have investigated a number of strategies to achieve the intracellular delivery of QDs including peptide-mediated, polymer-mediated, and microinjection based methods. In particular, the ability to selectively deliver biofunctionalized QDs to specific intracellular compartments is being targeted. Additionally, long-term QD intracellular QD fate, stability and toxicity are also concomitantly being examined. Cellular delivery experiments utilizing these various schemes will be highlighted and the relative advantages and disadvantages of each approach will be discussed.

  1. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria

    PubMed Central

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-01-01

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria. PMID:25009182

  2. Unveiling the Intracellular Survival Gene Kit of Trypanosomatid Parasites

    PubMed Central

    Bartholomeu, Daniella Castanheira; de Paiva, Rita Marcia Cardoso; Mendes, Tiago A. O.; DaRocha, Wanderson D.; Teixeira, Santuza M. R.

    2014-01-01

    Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas Trypanosoma cruzi and different species of Leishmania are obligate intracellular parasites, Trypanosoma brucei and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the T. cruzi or Leishmania genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different Trypanosoma and Leishmania strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism's mode of living and the associated disease phenotypes. Here, we review the studies concerning T. cruzi and Leishmania genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets. PMID:25474314

  3. Nanoparticles and intracellular applications of surface-enhanced Raman spectroscopy.

    PubMed

    Taylor, Jack; Huefner, Anna; Li, Li; Wingfield, Jonathan; Mahajan, Sumeet

    2016-08-15

    Surface-enhanced Raman spectrocopy (SERS) offers ultrasensitive vibrational fingerprinting at the nanoscale. Its non-destructive nature affords an ideal tool for interrogation of the intracellular environment, detecting the localisation of biomolecules, delivery and monitoring of therapeutics and for characterisation of complex cellular processes at the molecular level. Innovations in nanotechnology have produced a wide selection of novel, purpose-built plasmonic nanostructures capable of high SERS enhancement for intracellular probing while microfluidic technologies are being utilised to reproducibly synthesise nanoparticle (NP) probes at large scale and in high throughput. Sophisticated multivariate analysis techniques unlock the wealth of previously unattainable biomolecular information contained within large and multidimensional SERS datasets. Thus, with suitable combination of experimental techniques and analytics, SERS boasts enormous potential for cell based assays and to expand our understanding of the intracellular environment. In this review we trace the pathway to utilisation of nanomaterials for intracellular SERS. Thus we review and assess nanoparticle synthesis methods, their toxicity and cell interactions before presenting significant developments in intracellular SERS methodologies and how identified challenges can be addressed. PMID:27479539

  4. Caged ligands to study the role of intracellular GPCRs.

    PubMed

    Tadevosyan, Artavazd; Villeneuve, Louis R; Fournier, Alain; Chatenet, David; Nattel, Stanley; Allen, Bruce G

    2016-01-01

    In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48-60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface. PMID:26196333

  5. Community-acquired pneumonia related to intracellular pathogens.

    PubMed

    Cillóniz, Catia; Torres, Antoni; Niederman, Michael; van der Eerden, Menno; Chalmers, James; Welte, Tobias; Blasi, Francesco

    2016-09-01

    Community-acquired pneumonia (CAP) is associated with high rates of morbidity and mortality worldwide; the annual incidence of CAP among adults in Europe has ranged from 1.5 to 1.7 per 1000 population. Intracellular bacteria are common causes of CAP. However, there is considerable variation in the reported incidence between countries and change over time. The intracellular pathogens that are well established as causes of pneumonia are Legionella pneumophila, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Chlamydophila psittaci, and Coxiella burnetii. Since it is known that antibiotic treatment for severe CAP is empiric and includes coverage of typical and atypical pathogens, microbiological diagnosis bears an important relationship to prognosis of pneumonia. Factors such as adequacy of initial antibiotic or early de-escalation of therapy are important variables associated with outcomes, especially in severe cases. Intracellular pathogens sometimes appear to cause more severe disease with respiratory failure and multisystem dysfunction associated with fatal outcomes. The clinical relevance of intracellular pathogens in severe CAP has not been specifically investigated. We review the prevalence, general characteristics, and outcomes of severe CAP cases caused by intracellular pathogens. PMID:27276986

  6. Transport and intracellular accumulation of acetaldehyde in Saccharomyces cerevisiae

    SciTech Connect

    Stanley, G.A.; Pamment, N.B. )

    1993-06-05

    The rate of acetaldehyde efflux from yeast cells and its intracellular concentration were studied in the light of recent suggestions that acetaldehyde inhibition may be an important factor in yeast ethanol fermentations. When the medium surrounding cells containing ethanol and acetaldehyde was suddenly diluted, the rate of efflux of acetaldehyde was slow relative to the rate of ethanol efflux, suggesting that acetaldehyde, unlike ethanol, may accumulate intracellularly. Intracellular acetaldehyde concentrations were measured during high cell density fermentations, using direct injection gas chromatography to avoid the need to concentrate or disrupt the cells. Intracellular acetaldehyde concentrations substantially exceeded the extracellular concentrations throughout fermentation and were generally much higher than the acetaldehyde concentrations normally recorded in the culture broth in ethanol fermentations. The technique used was sensitive to the time taken to cool and freeze the samples. Measured intracellular acetaldehyde concentrations fell rapidly as the time taken to freeze the suspensions was extended beyond 2 s. The results add weight to recent claims that acetaldehyde toxicity is responsible for some of the effects previously ascribed to ethanol in alcohol fermentations, especially Zymomonas fermentations. Further work is required to confirm the importance of acetaldehyde toxicity under other culture conditions.

  7. Hijacking and Use of Host Lipids by Intracellular Pathogens.

    PubMed

    Toledo, Alvaro; Benach, Jorge L

    2015-12-01

    Intracellular bacteria use a number of strategies to survive, grow, multiply, and disseminate within the host. One of the most striking adaptations that intracellular pathogens have developed is the ability to utilize host lipids and their metabolism. Bacteria such as Anaplasma, Chlamydia, or Mycobacterium can use host lipids for different purposes, such as a means of entry through lipid rafts, building blocks for bacteria membrane formation, energy sources, camouflage to avoid the fusion of phagosomes and lysosomes, and dissemination. One of the most extreme examples of lipid exploitation is Mycobacterium, which not only utilizes the host lipid as a carbon and energy source but is also able to reprogram the host lipid metabolism. Likewise, Chlamydia spp. have also developed numerous mechanisms to reprogram lipids onto their intracellular inclusions. Finally, while the ability to exploit host lipids is important in intracellular bacteria, it is not an exclusive trait. Extracellular pathogens, including Helicobacter, Mycoplasma, and Borrelia, can recruit and metabolize host lipids that are important for their growth and survival.Throughout this chapter we will review how intracellular and extracellular bacterial pathogens utilize host lipids to enter, survive, multiply, and disseminate in the host. PMID:27337282

  8. Intracellular Neural Recording with Pure Carbon Nanotube Probes

    PubMed Central

    Yoon, Inho; Hamaguchi, Kosuke; Borzenets, Ivan V.; Finkelstein, Gleb; Mooney, Richard; Donald, Bruce R.

    2013-01-01

    The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications. PMID:23840357

  9. Magnesium relaxes arterial smooth muscle by decreasing intracellular Ca2+ without changing intracellular Mg2+.

    PubMed Central

    D'Angelo, E K; Singer, H A; Rembold, C M

    1992-01-01

    Elevations in extracellular [Mg2+] ([Mg2+]o) relax vascular smooth muscle. We tested the hypothesis that elevated [Mg2+]o induces relaxation through reductions in myoplasmic [Ca2+] and myosin light chain phosphorylation without changing intracellular [Mg2+] ([Mg2+]i). Histamine stimulation of endothelium-free swine carotid medial tissues was associated with increases in both Fura 2- and aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and force. Elevated [Mg2+]o decreased myoplasmic [Ca2+] and force to near resting values. However, elevated [Mg2+]o only transiently decreased myosin phosphorylation values: sustained [Mg2+]o-induced decreases in myoplasmic [Ca2+] and force were associated with inappropriately high myosin phosphorylation values. The elevated myosin phosphorylation during [Mg2+]o-induced relaxation was entirely on serine 19, the Ca2+/calmodulin-dependent myosin light chain kinase substrate. Myoplasmic [Mg2+] (estimated with Mag-Fura 2) did not significantly increase with elevated [Mg2+]o. These results are consistent with the hypothesis that increased [Mg2+]o induces relaxation by decreasing myoplasmic [Ca2+] without changing [Mg2+]i. These data also demonstrate dissociation of myosin phosphorylation from myoplasmic [Ca2+] and force during Mg(2+)-induced relaxation. This finding suggests the presence of a phosphorylation-independent (yet potentially Ca(2+)-dependent) mechanism for regulation of force in vascular smooth muscle. Images PMID:1602005

  10. Development of protein mimics for intracellular delivery.

    PubMed

    deRonde, Brittany M; Tew, Gregory N

    2015-07-01

    Designing delivery agents for therapeutics is an ongoing challenge. As treatments and desired cargoes become more complex, the need for improved delivery vehicles becomes critical. Excellent delivery vehicles must ensure the stability of the cargo, maintain the cargo's solubility, and promote efficient delivery and release. In order to address these issues, many research groups have looked to nature for design inspiration. Proteins, such as HIV-1 trans-activator of transcription (TAT) and Antennapedia homeodomain protein, are capable of crossing cellular membranes. However, due to the complexities of their structures, they are synthetically challenging to reproduce in the laboratory setting. Being able to incorporate the key features of these proteins that enable cell entry into simpler scaffolds opens up a wide range of opportunities for the development of new delivery reagents with improved performance. This review charts the development of protein mimics based on cell-penetrating peptides (CPPs) and how structure-activity relationships (SARs) with these molecules and their protein counterparts ultimately led to the use of polymeric scaffolds. These scaffolds deviate from the normal peptide backbone, allowing for simpler, synthetic procedures to make carriers and tune chemical compositions for application specific needs. Successful design of polymeric protein mimics would allow researchers to further understand the key features in proteins and peptides necessary for efficient delivery and to design the next generation of more efficient delivery reagents. PMID:25858701

  11. Vertical nanowire probes for intracellular signaling of living cells

    PubMed Central

    2014-01-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz. PMID:24484729

  12. Vertical nanowire probes for intracellular signaling of living cells

    NASA Astrophysics Data System (ADS)

    Lee, Ki-Young; Kim, Ilsoo; Kim, So-Eun; Jeong, Du-Won; Kim, Ju-Jin; Rhim, Hyewhon; Ahn, Jae-Pyeong; Park, Seung-Han; Choi, Heon-Jin

    2014-02-01

    The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz.

  13. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

    PubMed

    Lehar, Sophie M; Pillow, Thomas; Xu, Min; Staben, Leanna; Kajihara, Kimberly K; Vandlen, Richard; DePalatis, Laura; Raab, Helga; Hazenbos, Wouter L; Morisaki, J Hiroshi; Kim, Janice; Park, Summer; Darwish, Martine; Lee, Byoung-Chul; Hernandez, Hilda; Loyet, Kelly M; Lupardus, Patrick; Fong, Rina; Yan, Donghong; Chalouni, Cecile; Luis, Elizabeth; Khalfin, Yana; Plise, Emile; Cheong, Jonathan; Lyssikatos, Joseph P; Strandh, Magnus; Koefoed, Klaus; Andersen, Peter S; Flygare, John A; Wah Tan, Man; Brown, Eric J; Mariathasan, Sanjeev

    2015-11-19

    Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections. PMID:26536114

  14. Measuring intracellular motion in cancer cell using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Zam, Azhar; Kolios, Michael C.

    2016-03-01

    In this study, we demonstrate that OCT speckle decorrelation techniques can be used to probe intracellular motion in cancer cells. Spheroids and cell pellets were used as a model to probe intracellular motion. ZnCl2 was used to inhibit mitochondrial motion within the cells. The results reveal the changes in intracellular motion during the spheroid growth phase. Moreover, to modify the motion of mitochondria, cell pellet were exposed to ZnCl2, and agent known to o impair cellular energy production through inhibition of mitochondrial function. The speckle decorrelation time during the growth phase of spheroids decreased by 35 ms over 21 days and 25 ms during inhibition of mitochondrial motion 10 minutes after exposure to ZnCl2.

  15. Direct determination of the intracellular oxidation state of plutonium.

    PubMed

    Gorman-Lewis, Drew; Aryal, Baikuntha P; Paunesku, Tatjana; Vogt, Stefan; Lai, Barry; Woloschak, Gayle E; Jensen, Mark P

    2011-08-15

    Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1-μm(2) areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 h in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits were always consistent with tetravalent Pu even though the intracellular milieu is generally reducing. PMID:21755934

  16. Neuronal Recordings with Solid-Conductor Intracellular Nanoelectrodes (SCINEs)

    PubMed Central

    Angle, Matthew R.; Schaefer, Andreas T.

    2012-01-01

    Direct electrical recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms subserving neuronal computation. Existing intracellular recording techniques, however, limit the accuracy and duration of such measurements by changing intracellular biochemistry and/or by damaging the plasma membrane. Here we demonstrate that nanoengineered electrodes can be used to record neuronal transmembrane potentials in brain tissue without causing these physiological perturbations. Using focused ion beam milling, we have fabricated Solid-Conductor Intracellular NanoElectrodes (SCINEs), from conventional tungsten microelectrodes. SCINEs have tips that are <300 nm in diameter for several micrometers, but can be easily handled and can be inserted into brain tissue. Performing simultaneous whole-cell patch recordings, we show that SCINEs can record action potentials (APs) as well as slower, subthreshold neuronal potentials without altering cellular properties. These results show a key role for nanotechnology in the development of new electrical recording techniques in neuroscience. PMID:22905231

  17. Direct Determination of the Intracellular Oxidation State of Plutonium

    PubMed Central

    Gorman-Lewis, Drew; Aryal, Baikuntha P.; Paunesku, Tatjana; Vogt, Stefan; Lai, Barry; Woloschak, Gayle E.; Jensen, Mark P.

    2013-01-01

    Microprobe X-ray absorption near edge structure (μ-XANES) measurements were used to determine directly, for the first time, the oxidation state of intracellular plutonium in individual 0.1 μm2 areas within single rat pheochromocytoma cells (PC12). The living cells were incubated in vitro for 3 hours in the presence of Pu added to the media in different oxidation states (Pu(III), Pu(IV), and Pu(VI)) and in different chemical forms. Regardless of the initial oxidation state or chemical form of Pu presented to the cells, the XANES spectra of the intracellular Pu deposits was always consistent with tetravalent Pu even though the intracellular milieu is generally reducing. PMID:21755934

  18. Motor-driven intracellular transport powers bacterial gliding motility.

    PubMed

    Sun, Mingzhai; Wartel, Morgane; Cascales, Eric; Shaevitz, Joshua W; Mignot, Tâm

    2011-05-01

    Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility. PMID:21482768

  19. EFFECT OF TETRACYCLINES ON THE INTRACELLULAR AMINO ACIDS OF MOLDS.

    PubMed

    FREEMAN, B A; CIRCO, R

    1963-07-01

    Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed. PMID:14051820

  20. Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

    PubMed

    Ayar, A; Thatcher, N M; Zehavi, U; Trentham, D R; Scott, R H

    1998-01-01

    The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of

  1. Intracellular RNA cleavage by the hairpin ribozyme.

    PubMed Central

    Seyhan, A A; Amaral, J; Burke, J M

    1998-01-01

    Studies involving ribozyme-directed inactivation of targeted RNA molecules have met with mixed success, making clear the importance of methods to measure and optimize ribozyme activity within cells. The interpretation of biochemical assays for determining ribozyme activity in the cellular environment have been complicated by recent results indicating that hammerhead and hairpin ribozymes can cleave RNA following cellular lysis. Here, we report the results of experiments in which the catalytic activity of hairpin ribozymes is monitored following expression in mammalian cells, and in which post-lysis cleavage is rigorously excluded through a series of biochemical and genetic controls. Following transient transfection, self-processing transcripts containing active and inactive hairpin ribozymes together with cleavable and non-cleavable substrates were generated within the cytoplasm of mouse OST7-1 cells using T7 RNA polymerase. Unprocessed RNA and products ofintracellular cleavage were detected and analyzed using a primer-extension assay. Ribozyme-containing transcripts accumulated to a level of 4 x 10(4) copies per cell, and self-processing proceeded to an extent of >75% within cells. Cellular RNA processing was blocked by mutations within the ribozyme (G8A, G21U) or substrate (DeltaA-1) that, in vitro , eliminate cleavage without affecting substrate binding. In addition to self-processing activity, trans -cleavage reactions were supported by the ribozyme-containing product of the self-processing reaction, and by the ribozyme linked to the non-cleavable substrate analog. Ribozyme activity was present in extracts of cells expressing constructs with active ribozyme domains. These results provide direct biochemical evidence for the catalytic activity of the hairpin ribozyme in a cellular environment, and indicate that self-processing ribozyme transcripts may be well suited for cellular RNA-inactivation experiments. PMID:9671810

  2. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms

    PubMed Central

    Mansilla Pareja, Maria Eugenia; Colombo, Maria I.

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance. PMID:24137567

  3. Fluorogenic Substrate Detection of Viable Intracellular and Extracellular Pathogenic Protozoa

    NASA Astrophysics Data System (ADS)

    Jackson, Peter R.; Pappas, Michael G.; Hansen, Brian D.

    1985-01-01

    Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.

  4. Genetic systems for studying obligate intracellular pathogens: an update.

    PubMed

    Wood, David O; Wood, Raphael R; Tucker, Aimee M

    2014-02-01

    Rapid advancements in the genetic manipulation of obligate intracellular bacterial pathogens have been made over the past two years. In this paper we attempt to summarize the work published since 2011 that documents these exciting accomplishments. Although each genus comprising this diverse group of pathogens poses unique problems, requiring modifications of established techniques and the introduction of new tools, all appear amenable to genetic analysis. Significantly, the field is moving forward from a focus on the identification and development of genetic techniques to their application in addressing crucial questions related to mechanisms of bacterial pathogenicity and the requirements of obligate intracellular growth. PMID:24581687

  5. Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

    PubMed

    Zimmerman, Matthew C; Takapoo, Maysam; Jagadeesha, Dammanahalli K; Stanic, Bojana; Banfi, Botond; Bhalla, Ramesh C; Miller, Francis J

    2011-09-01

    Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration. PMID:21810651

  6. Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air-liquid interface

    SciTech Connect

    Mihai, Cosmin; Chrisler, William B.; Xie, Yumei; Hu, Dehong; Szymanski, Craig J.; Tolic, Ana; Klein, Jessica; Smith, Jordan N.; Tarasevich, Barbara J.; Orr, Galya

    2015-02-01

    Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn2+) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air-liquid interface (ALI). Using a fluorescent indicator for Zn2+, together with organelle-specific fluorescent proteins, we quantified Zn2+ in single cells and organelles over time. We found that at the ALI, intracellular Zn2+ values peaked 3 h post exposure and decayed to normal values by 12 h, while in submersed cultures, intracellular Zn2+ values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn2+ values that were nearly 3 folds lower than the peak values generated by the lowest toxic dose of NPs in submersed cultures, and 8 folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn2+. At the ALI, the majority of intracellular Zn2+ was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn2+ following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn2+ have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

  7. Intracellular hypertonicity is responsible for water flux associated with Na+/glucose cotransport.

    PubMed

    Charron, François M; Blanchard, Maxime G; Lapointe, Jean-Yves

    2006-05-15

    Detection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na(+)/glucose cotransporter (SGLT1) of two Na(+), one glucose, and 264 water molecules. Volumetric measurements of Xenopus laevis oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2 mM of the substrate alpha-methyl-glucose (alphaMG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60 s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077 nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2 mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3 mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na(+) injection and Na(+)-selective electrode, the intracellular diffusion coefficient for Na(+) was estimated at 0.29 +/- 0.03 x 10(-5) cm(2) s(-1). Using the effect of intracellular alphaMG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of alphaMG was estimated at 0.15 +/- 0.01 x 10(-5) cm(2) s(-1). Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of

  8. Tumor intracellular redox status and drug resistance--serendipity or a causal relationship?

    PubMed

    Pervaiz, Shazib; Clement, Marie-Veronique

    2004-01-01

    Reducing tumor load by therapeutic induction of cell death in the transformed phenotype is the desirable goal of most chemotherapeutic regimens. Despite the tremendous strides made in our understanding of mechanisms that endow tumor cells with the ability to evade execution signals, development of chemo-resistance is still a major obstacle in the successful management of the disease. A host of factors have been implicated in the acquisition of the resistant phenotype, such as activation of drug efflux pumps, overexpression of proteins that inhibit cell death, absence of critical members of the death circuitry, and selective loss of cell cycle checkpoints. Consequently, it is now well established that the process of carcinogenesis is not only a result of an increase in cells' proliferative capacity, but a product of increased proliferation and defective or diminished cell death signaling. To that end, one of the critical determinants of cellular response to exogenous stimuli is the cellular redox status. Intracellular generation of reactive oxygen species (ROS) is tightly regulated by the intrinsic anti-oxidant defense systems. Despite the conventional dogma that ROS are harmful to the cell, experimental evidence over the last decade or so bear witness to the fact that ROS also play an important role as signaling molecules in diverse physiological processes. Indeed, low levels of intracellular ROS have been linked to cellular proliferation and cell cycle progression, which provides an explanation for the pro-oxidant state invariably associated with the transformed phenotype. Coupled to that are recent observations implicating pro-oxidant intracellular milieu in tumor cells' resistance to cell death signals delivered through the cell surface receptor or upon exposure to chemotherapeutic drugs. These studies provide convincing evidence to support a direct or indirect role for intracellular superoxide anion in creating an intracellular milieu non-permissive for cell

  9. Intracellular delivery of peptides and siRNAs using microbubble enhanced focused ultrasound

    NASA Astrophysics Data System (ADS)

    Kinoshita, Manabu; Hynynen, Kullervo

    2006-05-01

    Bioactive substances such as peptides and nucleic acid based agents have attracted great attention for the next generation drug for various diseases. However, the greatest challenge for using these bioactive substances is the development of their delivery system, especially the method for delivering these substances through the cell membrane. With the advancement of ultrasound and ultrasound contrast agent technology, it has become possible to transiently change the permeability of the cell membrane. Moreover, using a focused ultrasound transducer, it is possible to narrow and focus the ultrasound energy within a small target, avoiding damage to the surrounding tissue. In this research we have searched the possibility of delivering the Bak BH3 peptide, the death domain of the Bc1-2 family of proteins, or the short interfering RNA (siRNA) targeting the enhanced green fluorescent protein (EGFP) using microbubble-enhanced focused ultrasound in an in vitro setting. Using a 1.696 MHz focused ultrasound and a microbubble ultrasound contrast agent OPTISON®, we first tested the stability of BH3 peptide under microbubble-enhanced focused ultrasound exposure and proved that the peptide is stable under these circumstances. Next, we have tested the cell-killing effect of the intracellularly delivered Bak BH3 peptide in HeLa and BJAB cell line and observed a statistically enhanced cell death in BJAB cells but not in HeLa cells, leading to the conclusion that intracellularly delivered BH3 peptide by microbubble-enhanced ultrasound can exert its cell killing effect in some cells. We also investigated if we can silence the EGFP expression in the cell by delivering siRNA targeting the EGFP in both transient and stable EGFP expression cell line. Using a 1.653 MHz focused ultrasound and OPTISON®, in both cases, intracellularly delivered siRNA by microbubble-enhanced ultrasound was able to knock down the EGFP expression, which demonstrates the feasibility of using this novel method

  10. Notch intracellular domain overexpression in adipocytes confers lipodystrophy in mice

    PubMed Central

    Chartoumpekis, Dionysios V.; Palliyaguru, Dushani L.; Wakabayashi, Nobunao; Khoo, Nicholas K.H.; Schoiswohl, Gabriele; O'Doherty, Robert M.; Kensler, Thomas W.

    2015-01-01

    Objective The Notch family of intermembrane receptors is highly conserved across species and is involved in cell fate and lineage control. Previous in vitro studies have shown that Notch may inhibit adipogenesis. Here we describe the role of Notch in adipose tissue by employing an in vivo murine model which overexpresses Notch in adipose tissue. Methods Albino C57BL/6J RosaNICD/NICD::Adipoq-Cre (Ad-NICD) male mice were generated to overexpress the Notch intracellular domain (NICD) specifically in adipocytes. Male RosaNICD/NICD mice were used as controls. Mice were evaluated metabolically at the ages of 1 and 3 months by assessing body weights, serum metabolites, body composition (EchoMRI), glucose tolerance and insulin tolerance. Histological sections of adipose tissue depots as well as of liver were examined. The mRNA expression profile of genes involved in adipogenesis was analyzed by quantitative real-time PCR. Results The Ad-NICD mice were heavier with significantly lower body fat mass compared to the controls. Small amounts of white adipose tissue could be seen in the 1-month old Ad-NICD mice, but was almost absent in the 3-months old mice. The Ad-NICD mice also had higher serum levels of glucose, insulin, triglyceride and non-esterified fatty acids. These differences were more prominent in the older (3-months) than in the younger (1-month) mice. The Ad-NICD mice also showed severe insulin resistance along with a steatotic liver. Gene expression analysis in the adipose tissue depots showed a significant repression of lipogenic (Fasn, Acacb) and adipogenic pathways (C/ebpα, C/ebpβ, Pparγ2, Srebf1). Conclusions Increased Notch signaling in adipocytes in mice results in blocked expansion of white adipose tissue which leads to ectopic accumulation of lipids and insulin resistance, thus to a lipodystrophic phenotype. These results suggest that further investigation of the role of Notch signaling in adipocytes could lead to the manipulation of this pathway for

  11. Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids.

    PubMed

    Kim, Jinsung; Moon, Sang Hui; Shin, Young-Cheul; Jeon, Ju-Hong; Park, Kyu Joo; Lee, Kyu Pil; So, Insuk

    2016-04-01

    Transient receptor potential canonical (TRPC) 4 channels are calcium-permeable, nonselective cation channels and are widely expressed in mammalian tissue, especially in the GI tract and brain. TRPC4 channels are known to be involved in neurogenic contraction of ileal smooth muscle cells via generating cationic current after muscarinic stimulation (muscarinic cationic current (mIcat)). Polyamines exist in numerous tissues and are believed to be involved in cell proliferation, differentiation, scar formation, wound healing, and carcinogenesis. Besides, physiological polyamines are essential to maintain inward rectification of cardiac potassium channels (Kir2.1). At membrane potentials more positive than equilibrium potential, intracellular polyamines plug the cytosolic surface of the Kir2.1 so that potassium ions cannot pass through the pore. Recently, it was reported that polyamines inhibit not only cardiac potassium channels but also nonselective cation channels that mediate the generation of mIcat. Here, we report that TRPC4, a definite mIcat mediator, is inhibited by intracellular spermine with great extent. The inhibition was specific to TRPC4 and TRPC5 channels but was not effective to TRPC1/4, TRPC1/5, and TRPC3 channels. For this inhibition to occur, we found that glutamates at 728th and 729th position of TRPC4 channels are essential whereby we conclude that spermine blocks the TRPC4 channel with electrostatic interaction between negative amino acids at the C-terminus of the channel. PMID:26631167

  12. pH-Responsive mineralized nanoparticles as stable nanocarriers for intracellular nitric oxide delivery.

    PubMed

    Lee, Hong Jae; Kim, Da Eun; Park, Dong Jin; Choi, Gi Hyun; Yang, Dal-Nim; Heo, Jung Sun; Lee, Sang Cheon

    2016-10-01

    We describe a calcium carbonate (CaCO3) mineralization approach to generate pH-responsive nanocarriers that can stably load S-nitrosoglutathione (GSNO) and dissolve at acidic endosomes to trigger intracellular release of nitric oxide (NO). GSNO-loaded CaCO3-mineralized nanoparticles (GSNO-MNPs) were prepared by an anionic block copolymer (PEG-Poly(l-aspartic acid))-templated mineralization. Ionic GSNO could be loaded in situ inside the CaCO3 core during the mineralization process. The stability of GSNO shielded within the crystalline CaCO3 core was greatly enhanced. The GSNO-MNPs triggered NO release at endosomal pH and an intracellular ascorbic acid level. Confocal microscopy demonstrated that the GSNO-MNPs could be dissolved at endosomal environments to release GSNO and sequentially generate NO through the GSNO reduction in the cytosol. In vitro cell experiments demonstrated that NO release by the GSNO-MNPs efficiently improved therapeutic activity of doxorubicin (DOX). PMID:27240199

  13. Protective role of intracellular glutathione against ethanol-induced damage in cultured rat gastric mucosal cells

    SciTech Connect

    Mutoh, H.; Hiraishi, H.; Ota, S.; Yoshida, H.; Ivey, K.J.; Terano, A.; Sugimoto, T. )

    1990-06-01

    This study investigated whether intracellular glutathione is cytoprotective against ethanol-induced injury to cultured rat gastric mucosal cells in vitro. Secondly, it investigated whether reduced glutathione or oxidized glutathione is responsible for this cytoprotection. Cytolysis was quantified by measuring 51Cr release from prelabeled cells. Concentrations of ethanol greater than 12% caused cell damage and increased 51Cr release in a dose-dependent and time-related fashion. When a substrate for glutathione synthesis, N-acetyl-L-cysteine, was provided to cultured cells for 4 h before challenge with ethanol, cytolysis was significantly decreased corresponding with an increase in cellular glutathione content. Pretreatment with diethyl maleate, which depletes reduced glutathione without forming oxidized glutathione, potentiated ethanol-induced cell damage in a dose-dependent manner with the decrease of cellular glutathione content. The administration of tert-butyl hydroperoxide (which is specifically reduced by glutathione peroxidase to generate oxidized glutathione from reduced glutathione) or diamide (which nonenzymatically oxidizes reduced glutathione to oxidized glutathione) enhanced ethanol injury. We conclude that in cultured gastric mucosal cells, (a) intracellular glutathione maintains integrity of gastric mucosal cells against ethanol in vitro; and (b) reduced glutathione rather than oxidized glutathione is responsible for this cytoprotection. We postulate that the presence of reduced glutathione is essential to allow glutathione peroxidase to catalyze the ethanol-generated toxic oxygen radical, hydrogen peroxide.

  14. Imaging atrial arrhythmic intracellular calcium in intact heart

    PubMed Central

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R.

    2014-01-01

    Abnormalities in intracellular Ca2+ signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca2+ in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca2+ imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca2+ activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca2+ waves and Ca2+ alternans. Moreover, we applied this strategy to analyze Ca2+ signals in the hearts of WT and knock-in mice harboring a ‘leaky’ type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca2+ leak increases the susceptibility to Ca2+ alternans and Ca2+ waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca2+ leak via RyR2 by acute treatment with S107 reduced both Ca2+ alternans and Ca2+ waves, and prevented atrial arrhythmias. PMID:24041536

  15. Deciphering the Intracellular Fate of Propionibacterium acnes in Macrophages

    PubMed Central

    Fischer, Natalie; Mak, Tim N.; Shinohara, Debika Biswal; Sfanos, Karen S.; Meyer, Thomas F.

    2013-01-01

    Propionibacterium acnes is a Gram-positive bacterium that colonizes various niches of the human body, particularly the sebaceous follicles of the skin. Over the last years a role of this common skin bacterium as an opportunistic pathogen has been explored. Persistence of P. acnes in host tissue has been associated with chronic inflammation and disease development, for example, in prostate pathologies. This study investigated the intracellular fate of P. acnes in macrophages after phagocytosis. In a mouse model of P. acnes-induced chronic prostatic inflammation, the bacterium could be detected in prostate-infiltrating macrophages at 2 weeks postinfection. Further studies performed in the human macrophage cell line THP-1 revealed intracellular survival and persistence of P. acnes but no intracellular replication or escape from the host cell. Confocal analyses of phagosome acidification and maturation were performed. Acidification of P. acnes-containing phagosomes was observed at 6 h postinfection but then lost again, indicative of cytosolic escape of P. acnes or intraphagosomal pH neutralization. No colocalization with the lysosomal markers LAMP1 and cathepsin D was observed, implying that the P. acnes-containing phagosome does not fuse with lysosomes. Our findings give first insights into the intracellular fate of P. acnes; its persistency is likely to be important for the development of P. acnes-associated inflammatory diseases. PMID:23862148

  16. Mitochondrial dysfunction and intracellular calcium dysregulation in ALS

    PubMed Central

    Kawamata, Hibiki; Manfredi, Giovanni

    2010-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that affects the aging population. A progressive loss of motor neurons in the spinal cord and brain leads to muscle paralysis and death. As in other common neurodegenerative diseases, aging-related mitochondrial dysfunction is increasingly being considered among the pathogenic factors. Mitochondria are critical for cell survival: they provide energy to the cell, buffer intracellular calcium, and regulate apoptotic cell death. Whether mitochondrial abnormalities are a trigger or a consequence of the neurodegenerative process and the mechanisms whereby mitochondrial dysfunction contributes to disease are not clear yet. Calcium homeostasis is a major function of mitochondria in neurons, and there is ample evidence that intracellular calcium is dysregulated in ALS. The impact of mitochondrial dysfunction on intracellular calcium homeostasis and its role in motor neuron demise are intriguing issues that warrants in depth discussion. Clearly, unraveling the causal relationship between mitochondrial dysfunction, calcium dysregulation, and neuronal death is critical for the understanding of ALS pathogenesis. In this review, we will outline the current knowledge of various aspects of mitochondrial dysfunction in ALS, with a special emphasis on the role of these abnormalities on intracellular calcium handling. PMID:20493207

  17. Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila

    PubMed Central

    Fonseca, Maris V.; Swanson, Michele S.

    2014-01-01

    The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication. PMID:24575391

  18. Dynamic Reorganization of Metabolic Enzymes into Intracellular Bodies

    PubMed Central

    O’Connell, Jeremy D.; Zhao, Alice; Ellington, Andrew D.; Marcotte, Edward M.

    2013-01-01

    Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci—such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis—to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality—and dysfunctionality—of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. PMID:23057741

  19. The proteome targets of intracellular targeting antimicrobial peptides.

    PubMed

    Shah, Pramod; Hsiao, Felix Shih-Hsiang; Ho, Yu-Hsuan; Chen, Chien-Sheng

    2016-04-01

    Antimicrobial peptides have been considered well-deserving candidates to fight the battle against microorganisms due to their broad-spectrum antimicrobial activities. Several studies have suggested that membrane disruption is the basic mechanism of AMPs that leads to killing or inhibiting microorganisms. Also, AMPs have been reported to interact with macromolecules inside the microbial cells such as nucleic acids (DNA/RNA), protein synthesis, essential enzymes, membrane septum formation and cell wall synthesis. Proteins are associated with many intracellular mechanisms of cells, thus protein targets may be specifically involved in mechanisms of action of AMPs. AMPs like pyrrhocoricin, drosocin, apidecin and Bac 7 are documented to have protein targets, DnaK and GroEL. Moreover, the intracellular targeting AMPs are reported to influence more than one protein targets inside the cell, suggesting for the multiple modes of actions. This complex mechanism of intracellular targeting AMPs makes them more difficult for the development of resistance. Herein, we have summarized the current status of AMPs in terms of their mode of actions, entry to cytoplasm and inhibition of macromolecules. To reveal the mechanism of action, we have focused on AMPs with intracellular protein targets. We have also included the use of high-throughput proteome microarray to determine the unidentified AMP protein targets in this review. PMID:26648572

  20. Intracellular GTP level determines cell's fate toward differentiation and apoptosis

    SciTech Connect

    Meshkini, Azadeh; Yazdanparast, Razieh Nouri, Kazem

    2011-06-15

    Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKC{alpha} and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis. - Graphical abstract: Display Omitted

  1. Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico

    PubMed Central

    Pierlé, Sebastián Aguilar; Morales, Cirani Obregón; Martínez, Leonardo Perea; Ceballos, Nidia Aréchiga; Rivero, Juan José Pérez; Díaz, Osvaldo López; Brayton, Kelly A.

    2015-01-01

    An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc. PMID:26583968

  2. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy

    EPA Science Inventory

    There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multip...

  3. Activity of 10 antimicrobial agents against intracellular Rhodococcus equi.

    PubMed

    Giguère, Steeve; Berghaus, Londa J; Lee, Elise A

    2015-08-01

    Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocyte-derived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. PMID:26051479

  4. Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

    PubMed

    Nguyen, Mai Khanh; Kim, Cha Yeon; Kim, Jin Man; Park, Byung Ouk; Lee, Sangkyu; Park, Hyerim; Heo, Won Do

    2016-06-01

    Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems. PMID:27065232

  5. Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico.

    PubMed

    Pierlé, Sebastián Aguilar; Morales, Cirani Obregón; Martínez, Leonardo Perea; Ceballos, Nidia Aréchiga; Rivero, Juan José Pérez; Díaz, Osvaldo López; Brayton, Kelly A; Setién, Alvaro Aguilar

    2015-12-01

    An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc. PMID:26583968

  6. Intracellular pH measurements using perfluorocarbon nanoemulsions.

    PubMed

    Patrick, Michael J; Janjic, Jelena M; Teng, Haibing; O'Hear, Meredith R; Brown, Cortlyn W; Stokum, Jesse A; Schmidt, Brigitte F; Ahrens, Eric T; Waggoner, Alan S

    2013-12-11

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time. PMID:24266634

  7. Intracellular pH measurements using perfluorocarbon nanoemulsions

    PubMed Central

    Patrick, Michael J.; Janjic, Jelena M.; Teng, Haibing; O’Hear, Meredith R.; Brown, Cortlyn W.; Stokum, Jesse A.; Schmidt, Brigitte F.; Ahrens, Eric T.; Waggoner, Alan S.

    2014-01-01

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon co-incubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions where characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry, and displayed a steady decrease in pH to a level of 5.5 over 3 hours, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using 19F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics and nanoemulsion stability and cell viability over time. PMID:24266634

  8. Caenorhabditis elegans as a model for intracellular pathogen infection

    PubMed Central

    Balla, Keir M.; Troemel, Emily R.

    2014-01-01

    Summary The genetically tractable nematode Caenorhabditis elegans is a convenient host for studies of pathogen infection. With the recent identification of two types of natural intracellular pathogens of C. elegans, this host now provides the opportunity to examine interactions and defence against intracellular pathogens in a whole-animal model for infection. C. elegans is the natural host for a genus of microsporidia, which comprise a phylum of fungal-related pathogens of widespread importance for agriculture and medicine. More recently, C. elegans has been shown to be a natural host for viruses related to the Nodaviridae family. Both microsporidian and viral pathogens infect the C. elegans intestine, which is composed of cells that share striking similarities to human intestinal epithelial cells. Because C. elegans nematodes are transparent, these infections provide a unique opportunity to visualize differentiated intestinal cells in vivo during the course of intracellular infection. Together, these two natural pathogens of C. elegans provide powerful systems in which to study microbial pathogenesis and host responses to intracellular infection. PMID:23617769

  9. Luciferase-based protein-denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: developing an intracellular thermometer

    PubMed Central

    Raoof, Mustafa; Zhu, Cihui; Kaluarachchi, Warna D.; Curley, Steven A.

    2013-01-01

    Background Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. Methods Water-bath or non-invasive external RF generator (600W, 13.56 MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and Propidium iodide staining. Heat shock proteins were analyzed using western-blot analysis Results Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water-bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4 minutes of RF exposure. Conclusions Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles. PMID:22515341

  10. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  11. Intracellular accumulation of azithromycin by cultured human fibroblasts.

    PubMed Central

    Gladue, R P; Snider, M E

    1990-01-01

    Azithromycin was shown to achieve high concentrations in human skin fibroblasts. Intracellular penetration occurred rapidly (10 micrograms/mg of cellular protein after 3 h) and then increased progressively over a 3-day period; azithromycin accumulated up to 21 times more than erythromycin (61.1 versus 2.9 micrograms/mg of protein). Uptake was dependent on the extracellular concentration, was inhibited at 4 degrees C, did not occur in nonviable cells, and was reduced by a low pH. Intracellular accumulation was not affected by the metabolic inhibitor 2,4-dinitrophenol or sodium fluoride or by the nucleoside transport inhibitor 2-chloradenosine. Once concentrated in cells, azithromycin remained intracellular and was released slowly in the absence of extracellular drug, compared with erythromycin (17 versus 78% released after 1 h). After 48 h of incubation in drug-free medium, 27% of the initial amount of azithromycin remained cell associated. The release of azithromycin was not affected by various monokines reported to stimulate fibroblasts (interleukin-1 or tumor necrosis factor) or by exposure to bacteria. Incubation of azithromycin-loaded fibroblasts with human polymorphonuclear leukocytes resulted in a higher intracellular accumulation of azithromycin in polymorphonuclear leukocytes than in cells incubated with free nonintracellular azithromycin for the same time (8.3 versus 2.2 micrograms/ml after 2 h), suggesting a more efficient or rapid uptake through cell-to-cell interaction. The widespread distribution of fibroblasts in tissues suggests a potential for these cells, and possibly other lysosome-containing tissue cells, to serve as a reservoir for azithromycin, slowly releasing it for activity against extracellular organisms at sites of infection and passing it to phagocytes for activity against intracellular pathogens and potential transport to sites of infection. PMID:2168141

  12. Intracellular Gene Expression Profile of Listeria monocytogenes †

    PubMed Central

    Chatterjee, Som Subhra; Hossain, Hamid; Otten, Sonja; Kuenne, Carsten; Kuchmina, Katja; Machata, Silke; Domann, Eugen; Chakraborty, Trinad; Hain, Torsten

    2006-01-01

    Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses. PMID:16428782

  13. Intracellular recording of action potentials by nanopillar electroporation

    NASA Astrophysics Data System (ADS)

    Xie, Chong; Lin, Ziliang; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

    2012-03-01

    Action potentials have a central role in the nervous system and in many cellular processes, notably those involving ion channels. The accurate measurement of action potentials requires efficient coupling between the cell membrane and the measuring electrodes. Intracellular recording methods such as patch clamping involve measuring the voltage or current across the cell membrane by accessing the cell interior with an electrode, allowing both the amplitude and shape of the action potentials to be recorded faithfully with high signal-to-noise ratios. However, the invasive nature of intracellular methods usually limits the recording time to a few hours, and their complexity makes it difficult to simultaneously record more than a few cells. Extracellular recording methods, such as multielectrode arrays and multitransistor arrays, are non-invasive and allow long-term and multiplexed measurements. However, extracellular recording sacrifices the one-to-one correspondence between the cells and electrodes, and also suffers from significantly reduced signal strength and quality. Extracellular techniques are not, therefore, able to record action potentials with the accuracy needed to explore the properties of ion channels. As a result, the pharmacological screening of ion-channel drugs is usually performed by low-throughput intracellular recording methods. The use of nanowire transistors, nanotube-coupled transistors and micro gold-spine and related electrodes can significantly improve the signal strength of recorded action potentials. Here, we show that vertical nanopillar electrodes can record both the extracellular and intracellular action potentials of cultured cardiomyocytes over a long period of time with excellent signal strength and quality. Moreover, it is possible to repeatedly switch between extracellular and intracellular recording by nanoscale electroporation and resealing processes. Furthermore, vertical nanopillar electrodes can detect subtle changes in action

  14. Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

    PubMed Central

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-01-01

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes. PMID:19071962

  15. Nonlinear Dynamic Modeling of Synaptically Driven Single Hippocampal Neuron Intracellular Activity

    PubMed Central

    Song, Dong; Berger, Theodore W.

    2011-01-01

    A high-order nonlinear dynamic model of the input–output properties of single hippocampal CA1 pyramidal neurons was developed based on synaptically driven intracellular activity. The purpose of this study is to construct a model that: 1) can capture the nonlinear dynamics of both subthreshold activities [postsynaptic potentials (PSPs)] and suprathreshold activities (action potentials) in a single formalism; 2) is sufficiently general to be applied to any spike-input and spike-output neurons (point process input and point process output neural systems); and 3) is computationally efficient. The model consisted of three major components: 1) feedforward kernels (up to third order) that transform presynaptic action potentials into PSPs; 2) a constant threshold, above which action potentials are generated; and 3) a feedback kernel (first order) that describes spike-triggered after-potentials. The model was applied to CA1 pyramidal cells, as they were electrically stimulated with broadband Poisson random impulse trains through the Schaffer collaterals. The random impulse trains used here have physiological properties similar to spiking patterns observed in CA3 hippocampal neurons. PSPs and action potentials were recorded from the soma of CA1 pyramidal neurons using whole-cell patch-clamp recording. We evaluated the model performance separately with respect to PSP waveforms and the occurrence of spikes. The average normalized mean square error of PSP prediction is 14.4%. The average spike prediction error rate is 18.8%. In summary, although prediction errors still could be reduced, the model successfully captures the majority of high-order nonlinear dynamics of the single-neuron intracellular activity. The model captures the general biophysical processes with a small set of open parameters that are directly constrained by the intracellular recording, and thus, can be easily applied to any spike-input and spike-output neuron. PMID:21233041

  16. Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model.

    PubMed

    Jenner, Dominic; Ducker, Catherine; Clark, Graeme; Prior, Jo; Rowland, Caroline A

    2016-04-01

    The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC. PMID:26841315

  17. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  18. Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza

    PubMed Central

    Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia, Junkal

    2016-01-01

    Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation. PMID:27124727

  19. Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

    PubMed

    Mell, Joshua Chang; Viadas, Cristina; Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A; St Geme, Joseph W; Nislow, Corey; Redfield, Rosemary J; Garmendia, Junkal

    2016-04-01

    Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation. PMID:27124727

  20. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

    PubMed

    Zhao, Zhenghang; Gordan, Richard; Wen, Hairuo; Fefelova, Nadezhda; Zang, Wei-Jin; Xie, Lai-Hua

    2013-01-01

    Recent studies have suggested that mitochondria may play important roles in the Ca(2+) homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+) flux can regulate the generation of Ca(2+) waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+) (Cai (2+)) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and CaWs were induced in the presence of high (4 mM) external Ca(2+) (Cao (2+)). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai (2+) levels even after depletion of SR Ca(2+) in the absence of Cao (2+) , suggesting Ca(2+) release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m ) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m ) or Ru360 (a mitochondrial Ca(2+) uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+) uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+) release and uptake exquisitely control the local Ca(2+) level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. PMID:24348912

  1. Analysing intracellular deformation of polymer capsules using structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

    2016-06-01

    Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces

  2. [Commemorative lecture of receiving Imamura Memorial Prize. Characterization of immunosuppressive macrophages induced in mice infected with Mycobacterium intracellulare].

    PubMed

    Tomioka, H

    1993-12-01

    Functional changes in T lymphocytes and macrophages (M phi s) in host mice during the course of Mycobacterium intracellulare infection were studied. In both strains of mice, BALB/c or C57BL/6 (susceptible to M. avium complex) and CBA/JN or C3H/He (resistant to M. avium complex), the smooth, opaque and dome-shaped colonial (SmD) variants of M. intracellulare were easily eliminated from the sites after week 2 of infection. In contrast, the smooth, transparent and irregularly shaped colonial (SmT) variants showed steady growth in the former strains of mice and persisted for long time even in the latter strains of mice. No difference was found between persistence of the organisms in euthymic (+/+) and athymic (nu/nu) BALB/c mice during the first 4 weeks after infection. Thereafter, more rapid growth was seen in the spleens and lungs of nu/nu mice. Thus, matured T cells may be important for the prevention of the progression of M. intracellulare infection to the terminal state. Next, the profiles of generation and characteristics of splenic M phi s which suppress the Con A mitogenic response of splenic T cells in host CBA/JN or BALB/c mice during the course of M. intracellulare infection were investigated. In M. intracellulare--infected mice, reduction in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after infection, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells (SPCs).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8301920

  3. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

    EPA Science Inventory

    Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

    ABSTRACT

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

  4. Intracellular protein degradation in mammalian cells: recent developments.

    PubMed

    Knecht, Erwin; Aguado, Carmen; Cárcel, Jaime; Esteban, Inmaculada; Esteve, Juan Miguel; Ghislat, Ghita; Moruno, José Félix; Vidal, José Manuel; Sáez, Rosana

    2009-08-01

    In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes. PMID:19399586

  5. Characterization of a Mycobacterium intracellulare Variant Strain by Molecular Techniques

    PubMed Central

    Menendez, M. C.; Palenque, E.; Navarro, M. C.; Nuñez, M. C.; Rebollo, M. J.; Garcia, M. J.

    2001-01-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members. PMID:11724827

  6. Evolution of the Calcium-Based Intracellular Signaling System.

    PubMed

    Marchadier, Elodie; Oates, Matt E; Fang, Hai; Donoghue, Philip C J; Hetherington, Alistair M; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  7. Highly potent intracellular membrane-associated Aβ seeds.

    PubMed

    Marzesco, Anne-Marie; Flötenmeyer, Matthias; Bühler, Anika; Obermüller, Ulrike; Staufenbiel, Matthias; Jucker, Mathias; Baumann, Frank

    2016-01-01

    An early event in Alzheimer's disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ. PMID:27311744

  8. Intracellular pH Modulates Autophagy and Mitophagy.

    PubMed

    Berezhnov, Alexey V; Soutar, Marc P M; Fedotova, Evgeniya I; Frolova, Maria S; Plun-Favreau, Helene; Zinchenko, Valery P; Abramov, Andrey Y

    2016-04-15

    The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control. PMID:26893374

  9. Monitoring the intracellular calcium response to a dynamic hypertonic environment

    NASA Astrophysics Data System (ADS)

    Huang, Xiaowen; Yue, Wanqing; Liu, Dandan; Yue, Jianbo; Li, Jiaqian; Sun, Dong; Yang, Mengsu; Wang, Zuankai

    2016-03-01

    The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening.

  10. Highly potent intracellular membrane-associated Aβ seeds

    PubMed Central

    Marzesco, Anne-Marie; Flötenmeyer, Matthias; Bühler, Anika; Obermüller, Ulrike; Staufenbiel, Matthias; Jucker, Mathias; Baumann, Frank

    2016-01-01

    An early event in Alzheimer’s disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ. PMID:27311744

  11. Multicomponent Supramolecular Polymers as a Modular Platform for Intracellular Delivery.

    PubMed

    Bakker, Maarten H; Lee, Cameron C; Meijer, E W; Dankers, Patricia Y W; Albertazzi, Lorenzo

    2016-02-23

    Supramolecular polymers are an emerging family of nanosized structures with potential use in materials chemistry and medicine. Surprisingly, application of supramolecular polymers in the field of drug delivery has received only limited attention. Here, we explore the potential of PEGylated 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for intracellular delivery. Exploiting the unique modular approach of supramolecular chemistry, we can coassemble neutral and cationic BTAs and control the overall properties of the polymer by simple monomer mixing. Moreover, this platform offers a versatile approach toward functionalization. The core can be efficiently loaded with a hydrophobic guest molecule, while the exterior can be electrostatically complexed with siRNA. It is demonstrated that both compounds can be delivered in living cells, and that they can be combined to enable a dual delivery strategy. These results show the advantages of employing a modular system and pave the way for application of supramolecular polymers in intracellular delivery. PMID:26811943

  12. Modulation of Host miRNAs by Intracellular Bacterial Pathogens.

    PubMed

    Das, Kishore; Garnica, Omar; Dhandayuthapani, Subramanian

    2016-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein coding genes of viruses and eukaryotes at the post-transcriptional level. The eukaryotic genes regulated by miRNAs include those whose products are critical for biological processes such as cell proliferation, metabolic pathways, immune response, and development. It is now increasingly recognized that modulation of miRNAs associated with biological processes is one of the strategies adopted by bacterial pathogens to survive inside host cells. In this review, we present an overview of the recent findings on alterations of miRNAs in the host cells by facultative intracellular bacterial pathogens. In addition, we discuss how the altered miRNAs help in the survival of these pathogens in the intracellular environment. PMID:27536558

  13. Pico gauges for minimally invasive intracellular hydrostatic pressure measurements.

    PubMed

    Knoblauch, Jan; Mullendore, Daniel L; Jensen, Kaare H; Knoblauch, Michael

    2014-11-01

    Intracellular pressure has a multitude of functions in cells surrounded by a cell wall or similar matrix in all kingdoms of life. The functions include cell growth, nastic movements, and penetration of tissue by parasites. The precise measurement of intracellular pressure in the majority of cells, however, remains difficult or impossible due to their small size and/or sensitivity to manipulation. Here, we report on a method that allows precise measurements in basically any cell type over all ranges of pressure. It is based on the compression of nanoliter and picoliter volumes of oil entrapped in the tip of microcapillaries, which we call pico gauges. The production of pico gauges can be accomplished with standard laboratory equipment, and measurements are comparably easy to conduct. Example pressure measurements are performed on cells that are difficult or impossible to measure with other methods. PMID:25232014

  14. Loligomers: design of de novo peptide-based intracellular vehicles.

    PubMed Central

    Sheldon, K; Liu, D; Ferguson, J; Gariépy, J

    1995-01-01

    Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents. Images Fig. 3 PMID:7892224

  15. On the structural organization of the intracellular domains of CFTR.

    PubMed

    Moran, Oscar

    2014-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein forming an anion selective channel. Mutations in the gene encoding CFTR cause cystic fibrosis (CF). The intracellular side of CFTR constitutes about 80% of the total mass of the protein. This region includes domains involved in ATP-dependent gating and regulatory protein kinase-A phosphorylation sites. The high-resolution molecular structure of CFTR has not yet been solved. However, a range of lower resolution structural data, as well as functional biochemical and electrophysiological data, are now available. This information has enabled the proposition of a working model for the structural architecture of the intracellular domains of the CFTR protein. PMID:24513531

  16. Non-contact intracellular binding of chloroplasts in vivo

    NASA Astrophysics Data System (ADS)

    Li, Yuchao; Xin, Hongbao; Liu, Xiaoshuai; Li, Baojun

    2015-06-01

    Non-contact intracellular binding and controllable manipulation of chloroplasts in vivo was demonstrated using an optical fiber probe. Launching a 980-nm laser beam into a fiber, which was placed about 3 μm above the surface of a living plant (Hydrilla verticillata) leaf, enabled stable binding of different numbers of chloroplasts, as well as their arrangement into one-dimensional chains and two-dimensional arrays inside the leaf without damaging the chloroplasts. Additionally, the formed chloroplast chains were controllably transported inside the living cells. The optical force exerted on the chloroplasts was calculated to explain the experimental results. This method provides a flexible method for studying intracellular organelle interaction with highly organized organelle-organelle contact in vivo in a non-contact manner.

  17. A Dual Wavelength Microfluorimeter for Measuring Fast Intracellular Calcium Signals

    NASA Astrophysics Data System (ADS)

    Hogan, Perry M.; Besch, Stephen R.

    1995-06-01

    A dual excitation microfluorimeter is described for measuring rapidly changing, intracellular calcium signals. A spinning sector wheel is used in conjunction with a beam masking device to provide rapid, efficient switching between the 2 excitation wavelengths. Exposure intervals as short as 120 [mu]s can be achieved, yielding ratio samples at a rate of 6 kHz. Emission photons are collected using a photomultiplier tube operating in counting mode. When tested using FURA-2 as the calcium reporting dye, throughput noise in the system is demonstrated to be due to the statistical fluctuation inherent in photon counting. An example of the operation of the system, using a guinea pig cardiac myocyte, demonstrates that sufficient ratio data may be acquires to fully characterize the fastest components of the intracellular calcium signal.

  18. Modulation of Host miRNAs by Intracellular Bacterial Pathogens

    PubMed Central

    Das, Kishore; Garnica, Omar; Dhandayuthapani, Subramanian

    2016-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein coding genes of viruses and eukaryotes at the post-transcriptional level. The eukaryotic genes regulated by miRNAs include those whose products are critical for biological processes such as cell proliferation, metabolic pathways, immune response, and development. It is now increasingly recognized that modulation of miRNAs associated with biological processes is one of the strategies adopted by bacterial pathogens to survive inside host cells. In this review, we present an overview of the recent findings on alterations of miRNAs in the host cells by facultative intracellular bacterial pathogens. In addition, we discuss how the altered miRNAs help in the survival of these pathogens in the intracellular environment. PMID:27536558

  19. Evolution of the Calcium-Based Intracellular Signaling System

    PubMed Central

    Marchadier, Elodie; Oates, Matt E.; Fang, Hai; Donoghue, Philip C.J.; Hetherington, Alistair M.; Gough, Julian

    2016-01-01

    To progress our understanding of molecular evolution from a collection of well-studied genes toward the level of the cell, we must consider whole systems. Here, we reveal the evolution of an important intracellular signaling system. The calcium-signaling toolkit is made up of different multidomain proteins that have undergone duplication, recombination, sequence divergence, and selection. The picture of evolution, considering the repertoire of proteins in the toolkit of both extant organisms and ancestors, is radically different from that of other systems. In eukaryotes, the repertoire increased in both abundance and diversity at a far greater rate than general genomic expansion. We describe how calcium-based intracellular signaling evolution differs not only in rate but in nature, and how this correlates with the disparity of plants and animals. PMID:27358427

  20. Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism

    PubMed Central

    Zubair, Haseeb; Azim, Shafquat; Khan, Husain Yar; Ullah, Mohammad Fahad; Wu, Daocheng; Singh, Ajay Pratap; Hadi, Sheikh Mumtaz; Ahmad, Aamir

    2016-01-01

    There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach. PMID:27331811

  1. Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism.

    PubMed

    Zubair, Haseeb; Azim, Shafquat; Khan, Husain Yar; Ullah, Mohammad Fahad; Wu, Daocheng; Singh, Ajay Pratap; Hadi, Sheikh Mumtaz; Ahmad, Aamir

    2016-01-01

    There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach. PMID:27331811

  2. Eimeria tenella: parasite-specific incorporation of /sup 3/H-uracil as a quantitative measure of intracellular development

    SciTech Connect

    Schmatz, D.M.; Crane, M.S.; Murray, P.K.

    1986-02-01

    An assay has been developed using parasite-specific incorporation of /sup 3/H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, /sup 3/H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional /sup 3/H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of /sup 3/H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.

  3. Electrical slow waves in the mouse oviduct are dependent on extracellular and intracellular calcium sources

    PubMed Central

    Dixon, Rose Ellen; Britton, Fiona C.; Baker, Salah A.; Hennig, Grant W.; Rollings, Christina M.; Sanders, Kenton M.

    2011-01-01

    Spontaneous contractions of the myosalpinx are critical for oocyte transport along the oviduct. Slow waves, the electrical events that underlie myosalpinx contractions, are generated by a specialized network of pacemaker cells called oviduct interstitial cells of Cajal (ICC-OVI). The ionic basis of oviduct pacemaker activity is unknown. Intracellular recordings and Ca2+ imaging were performed to examine the role of extracellular and intracellular Ca2+ sources in slow wave generation. RT-PCR was performed to determine the transcriptional expression of Ca2+ channels. Molecular studies revealed most isoforms of L- and T-type calcium channels (Cav1.2,1.3,1.4,3.1,3.2,3.3) were expressed in myosalpinx. Reduction of extracellular Ca2+ concentration ([Ca2+]o) resulted in the abolition of slow waves and myosalpinx contractions without significantly affecting resting membrane potential (RMP). Spontaneous Ca2+ waves spread through ICC-OVI cells at a similar frequency to slow waves and were inhibited by reduced [Ca2+]o. Nifedipine depolarized RMP and inhibited slow waves; however, pacemaker activity returned when the membrane was repolarized with reduced extracellular K+ concentration ([K+]o). Ni2+ also depolarized RMP but failed to block slow waves. The importance of ryanodine and inositol 1,4,5 trisphosphate-sensitive stores were examined using ryanodine, tetracaine, caffeine, and 2-aminoethyl diphenylborinate. Results suggest that although both stores are involved in regulation of slow wave frequency, neither are exclusively essential. The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid inhibited pacemaker activity and Ca2+ waves suggesting that a functional SERCA pump is necessary for pacemaker activity. In conclusion, results from this study suggest that slow wave generation in the oviduct is voltage dependent, occurs in a membrane potential window, and is dependent on extracellular calcium and functional SERCA pumps. PMID:21881003

  4. Enhanced singlet oxygen production by photodynamic therapy and a novel method for its intracellular measurement.

    PubMed

    Pena Luengas, Sandra L; Marin, Gustavo Horacio; Aviles, Kevin; Cruz Acuña, Ricardo; Roque, Gustavo; Rodríguez Nieto, Felipe; Sanchez, Francisco; Tarditi, Adrián; Rivera, Luis; Mansilla, Eduardo

    2014-12-01

    The generation of singlet oxygen (SO) in the presence of specific photosensitizers (PSs) or semiconductor quantum dots (QDs) and its application in photodynamic therapy (PDT) is of great interest to develop cancer therapies with no need of surgery, chemotherapy, and/or radiotherapy. This work was focused on the identification of the main factors leading to the enhancement of SO production using Rose Bengal (RB), and Methylene Blue (MB) as PS species in organic and aqueous mediums. Subsequently, the capacity of zinc oxide (ZnO), zinc sulfide (ZnS), and ZnO/ZnS core-shell QDs as well as manganese (Mn(+2)) doped ZnO and ZnS nanoparticles (NPs) as potential PS was also investigated. Many variable parameters such as type of quencher, PSs, NPs, as well as its different concentrations, light source, excitation wavelength, reaction time, distance from light source, and nature of solvent were used. The degradation kinetics of the quenchers generated by SO species and the corresponding quantum yields were determined by monitoring the photo-oxidation of the chemical quencher and measuring its disappearance by fluorometry and spectrophotometry in the presence of NPs. Small intracellular changes of SO induced by these metal Zn (zinc) NPs and PDT could execute and accelerate deadly programs in these leukemic cells, providing in this way an innovative modality of treatment. In order to perform further more specific in vitro cytotoxic studies on B-chronic lymphocytic leukemia cells exposed to Zn NPs and PDT, we needed first to measure and ascertain those possible intracellular SO variations generated by this type of treatment; for this purpose, we have also developed and tested a novel method first described by us. PMID:25490599

  5. Enhanced Singlet Oxygen Production by Photodynamic Therapy and a Novel Method for Its Intracellular Measurement

    PubMed Central

    Marin, Gustavo Horacio; Aviles, Kevin; Acuña, Ricardo Cruz; Roque, Gustavo; Nieto, Felipe Rodríguez; Sanchez, Francisco; Tarditi, Adrián; Rivera, Luis; Mansilla, Eduardo

    2014-01-01

    Abstract The generation of singlet oxygen (SO) in the presence of specific photosensitizers (PSs) or semiconductor quantum dots (QDs) and its application in photodynamic therapy (PDT) is of great interest to develop cancer therapies with no need of surgery, chemotherapy, and/or radiotherapy. This work was focused on the identification of the main factors leading to the enhancement of SO production using Rose Bengal (RB), and Methylene Blue (MB) as PS species in organic and aqueous mediums. Subsequently, the capacity of zinc oxide (ZnO), zinc sulfide (ZnS), and ZnO/ZnS core-shell QDs as well as manganese (Mn+2) doped ZnO and ZnS nanoparticles (NPs) as potential PS was also investigated. Many variable parameters such as type of quencher, PSs, NPs, as well as its different concentrations, light source, excitation wavelength, reaction time, distance from light source, and nature of solvent were used. The degradation kinetics of the quenchers generated by SO species and the corresponding quantum yields were determined by monitoring the photo-oxidation of the chemical quencher and measuring its disappearance by fluorometry and spectrophotometry in the presence of NPs. Small intracellular changes of SO induced by these metal Zn (zinc) NPs and PDT could execute and accelerate deadly programs in these leukemic cells, providing in this way an innovative modality of treatment. In order to perform further more specific in vitro cytotoxic studies on B-chronic lymphocytic leukemia cells exposed to Zn NPs and PDT, we needed first to measure and ascertain those possible intracellular SO variations generated by this type of treatment; for this purpose, we have also developed and tested a novel method first described by us. PMID:25490599

  6. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    PubMed

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes. PMID:27516189

  7. Intracellular accumulation of boceprevir according to plasma concentrations and pharmacogenetics.

    PubMed

    Cusato, Jessica; Allegra, Sarah; De Nicolò, Amedeo; Boglione, Lucio; Fatiguso, Giovanna; Abdi, Adnan Mohamed; Cariti, Giuseppe; Di Perri, Giovanni; D'Avolio, Antonio

    2015-06-01

    Boceprevir (BOC) is a directly-acting antiviral agent for the treatment of hepatitis C virus genotype 1 (HCV-1) infection. It is a mixture of two stereoisomers, the inactive R and the active S isomers. No data have previously been published on BOC intracellular accumulation. In this study, BOC isomer concentrations in peripheral blood mononuclear cells (PBMCs) and plasma were determined. The influence of various single nucleotide polymorphisms (SNPs) on plasma and intracellular drug exposure at Week 4 of triple therapy were also evaluated. Plasma and intracellular BOC concentrations were determined at the end of the dosing interval (C(trough)) using a UPLC-MS/MS validated method. Allelic discrimination was performed through real-time PCR. Median plasma concentrations were 65.97 ng/mL for the S isomer and 36.31 ng/mL for the R isomer; the median S/R plasma concentration ratio was 1.66. The median PBMC concentration was 2285.88 ng/mL for the S isomer; the R isomer was undetectable within PBMCs. The median S isomer PBMC/plasma concentration ratio was 28.59. A significant positive correlation was found between plasma and PBMC S isomer concentrations. ABCB1 1236, SLC28A2 124 and IL28B rs12979860 SNPs were associated with the S isomer PBMC/plasma concentration ratio. In regression models, S isomer plasma levels and FokI polymorphism were able to predict S isomer intracellular exposure, whereas SNPs in AKR1, BCRP1 and SLC28A2 predicted the S isomer PBMC/plasma concentration ratio. No similar data regarding BOC pharmacogenetics and pharmacokinetics have been published previously. This study adds a novel and useful overview of the pharmacological properties of this drug. PMID:25836019

  8. Oxidative stress and intracellular infections: more iron to the fire.

    PubMed

    Andrews, Norma W

    2012-07-01

    The immune system's battle against pathogens includes the "respiratory burst," a rapid release of ROS from leukocytes, thought to play a role in destroying the invading species. In this issue of the JCI, Paiva et al. demonstrate that oxidative stress actually enhances infection with the protozoan Trypanosoma cruzi, by a mechanism that may involve facilitating parasite access to iron. Their findings suggest a novel direction for the development of drugs against intracellular parasites. PMID:22728929

  9. Uric acid utilization by Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates.

    PubMed Central

    Falkinham, J O; George, K L; Parker, B C; Gruft, H

    1983-01-01

    Forty-nine human and environmental isolates of Mycobacterium intracellulare and Mycobacterium scrofulaceum were tested for their ability to grow on uric acid and a number of its degradation products. Nearly all (88 to 90%) strains used uric acid or allantoin as a sole nitrogen source; fewer (47 to 69%) used allantoate, urea, or possibly ureidoglycollate. Enzymatic activities of one representative isolate demonstrated the existence of a uric acid degradation pathway resembling that in other aerobic microorganisms. PMID:6863220

  10. Reconstitution of intracellular environments in vitro and in artificial cells

    PubMed Central

    Fujiwara, Kei; Yanagisawa, Miho; Nomura, Shin-ichiro M.

    2014-01-01

    Toward reconstitution of living cells by artificial cells technology, it is critical process to understand the differences between mixtures of biomolecules and living cells. For the aim, we have developed procedures for preparation of an additive-free cell extract (AFCE) and for concentrating biomacromolecules in artificial cells. In this review, we introduce our recent progress to reconstitute intracellular environments in vitro and in artificial cells. PMID:27493497

  11. Chelation of intracellular calcium blocks insulin action in the adipocyte

    SciTech Connect

    Pershadsingh, H.A.; Shade, D.L.; Delfert, D.M.; McDonald, J.M.

    1987-02-01

    The hypothesis that intracellular Ca/sup 2 +/ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca/sup 2 +/ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl/sub 2/ and the Ca/sup 2 +/ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl/sub 2/ from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation and the ability of insulin to inhibit cAMP-stimulated lipolysis without affecting their basal activities. Incubation of cells with 100 ..mu..M quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of /sup 125/I=labeled insulin to adipocytes. These findings suggest that intracellular Ca/sup 2 +/ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca/sup 2 +/-dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin.

  12. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  13. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton

    PubMed Central

    LI, JIEJING; LI, YANXI; ZHANG, PENGHUI; NIU, HUA; SHI, YU

    2014-01-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F-actin cytoskeleton systems and resulted in β-tubulin degradation in an ubiquitin-proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F-actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F-actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  14. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton.

    PubMed

    Li, Jiejing; Li, Yanxi; Zhang, Penghui; Niu, Hua; Shi, Yu

    2014-12-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F‑actin cytoskeleton systems and resulted in β‑tubulin degradation in an ubiquitin‑proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F‑actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F‑actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  15. Intracellular replication is essential for the virulence of Salmonella typhimurium.

    PubMed

    Leung, K Y; Finlay, B B

    1991-12-15

    Salmonella typhimurium is a facultative intracellular parasite, capable of penetrating, surviving, and multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. We have been studying intracellular replication of S. typhimurium and found that it is essential in the pathogenesis of this bacterium. A total of 45,000 independent mini-Mu MudJ transposon mutants in S. typhimurium SL1344 were screened in Madin-Darby canine kidney (MDCK) epithelial cells with a beta-lactam, cefotaxime, to enrich for mutants defective for intracellular replication. Ten different auxotrophic (purine, pyrimidine, purine/methionine, and valine/isoleucine) and three prototrophic replication-defective mutants (Rep-) were identified. All Rep- mutants showed no differences in aerobic and anaerobic growth patterns, motility, serum sensitivity, mouse macrophage survival, iron uptake, and phosphate requirements. All Rep- mutants were unable to multiply inside MDCK, HeLa, and Caco-2 epithelial cells. When required nutrients for various auxotrophs were supplemented, auxotrophs then replicated inside MDCK cells. Although the parental strain multiplies in large vacuoles inside MDCK cells that distort the host cells, MDCK cells infected with the Rep- mutants appeared relatively normal and few bacteria were seen inside vacuoles. The purine auxotrophs and the three prototrophic Rep- mutants were highly attenuated in mice, and oral and intraperitoneal LD50 levels were 3 to 4 orders of magnitude higher than the wild type level. The three prototrophs were invasive and persisted in the murine organs such as livers and spleens for at least 3 weeks. Therefore, these prototrophic genes are needed for intracellular replication and are essential to the virulence of S. typhimurium. PMID:1763061

  16. Evaluation of two novel methods for assessing intracellular oxygen

    NASA Astrophysics Data System (ADS)

    Williams, Catrin F.; Kombrabail, M.; Vijayalakshmi, K.; White, Nick; Krishnamoorthy, G.; Lloyd, David

    2012-08-01

    The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti-sapphire Tsunami lasers (710-1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1-2 × 104 counts s-1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime

  17. Coexistence of amplitude and frequency modulations in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    de Pittà, Maurizio; Volman, Vladislav; Levine, Herbert; Pioggia, Giovanni; de Rossi, Danilo; Ben-Jacob, Eshel

    2008-03-01

    The complex dynamics of intracellular calcium regulates cellular responses to information encoded in extracellular signals. Here we study the encoding of these external signals in the context of the Li-Rinzel model. We show that by control of biophysical parameters the information can be encoded in amplitude modulation (AM), frequency modulation (FM), or mixed (AM and FM) modulation. We briefly discuss the possible implications of this role of information encoding for astrocytes.

  18. Intracellular proton access in a Cl(-)/H(+) antiporter.

    PubMed

    Lim, Hyun-Ho; Shane, Tania; Miller, Christopher

    2012-01-01

    Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H(+)-gated Cl(-) channels and Cl(-)/H(+) antiporters. Transmembrane H(+) movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl(-) ion pathway through these proteins, but the H(+) pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H(+) in its path through the protein. The extracellular-facing H(+) transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu(in), is buried within the protein. Two regions, denoted "polar" and "interfacial," at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu(in). Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl(-)/H(+) antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu(in) physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu(in). PMID:23239938

  19. [Experimental studies on allergenic effect of Mycobacterium intracellulare in cattle].

    PubMed

    Schulz, G

    1975-01-01

    Mycobacterium intracellulare, isolated from sawdust used as litter, was inoculated into 25 bullocks. Six developed a doubtful reaction to mammalian tuberculin and the remainder stayed negative. With avian tuberculin, 8 became positive, 10 doubtful and 7 remained negative. Using the interpretation key described by Goetze, Lauterbach and Nassal, the reactions were shown to be para-allergic. At slaughter there was no evidence of tuberculous lesions. PMID:1200752

  20. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    PubMed

    Sharma, Onkar; O'Seaghdha, Maghnus; Velarde, Jorge J; Wessels, Michael R

    2016-03-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  1. Engineering cells with intracellular agent-loaded microparticles to control cell phenotype.

    PubMed

    Ankrum, James A; Miranda, Oscar R; Ng, Kelvin S; Sarkar, Debanjan; Xu, Chenjie; Karp, Jeffrey M

    2014-02-01

    Cell therapies enable unprecedented treatment options to replace tissues, destroy tumors and facilitate regeneration. The greatest challenge facing cell therapy is the inability to control the fate and function of cells after transplantation. We have developed an approach to control cell phenotype in vitro and after transplantation by engineering cells with intracellular depots that continuously release phenotype-altering agents for days to weeks. The platform enables control of cells' secretome, viability, proliferation and differentiation, and the platform can be used to deliver drugs or other factors (e.g., dexamethasone, rhodamine and iron oxide) to the cell's microenvironment. The preparation, efficient internalization and intracellular stabilization of ∼1-μm drug-loaded microparticles are critical for establishing sustained control of cell phenotype. Herein we provide a protocol to generate and characterize micrometer-sized agent-doped poly(lactic-co-glycolic) acid (PLGA) particles by using a single-emulsion evaporation technique (7 h), to uniformly engineer cultured cells (15 h), to confirm particle internalization and to troubleshoot commonly experienced obstacles. PMID:24407352

  2. An integrative approach to understanding microbial diversity: from intracellular mechanisms to community structure

    PubMed Central

    Gudelj, Ivana; Weitz, Joshua S; Ferenci, Tom; Claire Horner-Devine, M; Marx, Christopher J; Meyer, Justin R; Forde, Samantha E

    2010-01-01

    Trade-offs have been put forward as essential to the generation and maintenance of diversity. However, variation in trade-offs is often determined at the molecular level, outside the scope of conventional ecological inquiry. In this study, we propose that understanding the intracellular basis for trade-offs in microbial systems can aid in predicting and interpreting patterns of diversity. First, we show how laboratory experiments and mathematical models have unveiled the hidden intracellular mechanisms underlying trade-offs key to microbial diversity: (i) metabolic and regulatory trade-offs in bacteria and yeast; (ii) life-history trade-offs in bacterial viruses. Next, we examine recent studies of marine microbes that have taken steps toward reconciling the molecular and the ecological views of trade-offs, despite the challenges in doing so in natural settings. Finally, we suggest avenues for research where mathematical modelling, experiments and studies of natural microbial communities provide a unique opportunity to integrate studies of diversity across multiple scales. PMID:20576029

  3. Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

    PubMed

    Vanaporn, Muthita; Wand, Matthew; Michell, Stephen L; Sarkar-Tyson, Mitali; Ireland, Philip; Goldman, Stan; Kewcharoenwong, Chidchamai; Rinchai, Darawan; Lertmemongkolchai, Ganjana; Titball, Richard W

    2011-08-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide. PMID:21659326

  4. Intracellular Staphylococcus aureus Escapes the Endosome and Induces Apoptosis in Epithelial Cells

    PubMed Central

    Bayles, Kenneth W.; Wesson, Carla A.; Liou, Linda E.; Fox, Lawrence K.; Bohach, Gregory A.; Trumble, W. R.

    1998-01-01

    We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes. PMID:9423876

  5. Regulation of Intracellular Structural Tension by Talin in the Axon Growth and Regeneration.

    PubMed

    Dingyu, Wang; Fanjie, Meng; Zhengzheng, Ding; Baosheng, Huang; Chao, Yang; Yi, Pan; Huiwen, Wu; Jun, Guo; Gang, Hu

    2016-09-01

    Intracellular tension is the most important characteristic of neuron polarization as well as the growth and regeneration of axons, which can be generated by motor proteins and conducted along the cytoskeleton. To better understand this process, we created Förster resonance energy transfer (FRET)-based tension probes that can be incorporated into microfilaments to provide a real-time measurement of forces in neuron cytoskeletons. We found that our probe could be used to assess the structural tension of neuron polarity. Nerve growth factor (NGF) upregulated structural forces, whereas the glial-scar inhibitors chondroitin sulfate proteoglycan (CSPG) and aggrecan weakened such forces. Notably, the tension across axons was distributed uniformly and remarkably stronger than that in the cell body in NGF-stimulated neurons. The mechanosensors talin/vinculin could antagonize the effect of glial-scar inhibitors via structural forces. However, E-cadherin was closely associated with glial-scar inhibitor-induced downregulation of structural forces. Talin/vinculin was involved in the negative regulation of E-cadherin transcription through the nuclear factor-kappa B pathway. Collectively, this study clarified the mechanism underlying intracellular tension in the growth and regeneration of axons which, conversely, can be regulated by talin and E-cadherin. PMID:26298665

  6. Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

    PubMed Central

    Ramos, Cathy I.; Igiesuorobo, Oghomwen; Wang, Qi; Serpe, Mihaela

    2015-01-01

    The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. PMID:25905467

  7. Intracellular Protein Shuttling: A Mechanism Relevant for Myelin Repair in Multiple Sclerosis?

    PubMed

    Göttle, Peter; Küry, Patrick

    2015-01-01

    A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. PMID:26151843

  8. Magneto-optical cellular chip model for intracellular orientational-dynamic-activity detection

    NASA Astrophysics Data System (ADS)

    Miyashita, Y.; Iwasaka, M.; Kurita, S.; Owada, N.

    2012-04-01

    In the present study, a magneto-optical cellular chip model (MoCCM) was developed to detect intracellular dynamics in macromolecules by using magneto-optical effects. For the purpose of cell-measurement under strong static magnetic fields of up to 10 T, we constructed a cellular chip model, which was a thin glass plate with a well for a cell culture. A cell line of osteoblast MC3T3-E1 was incubated in the glass well, and the well, 0.3 mm in depth, was sealed by a cover glass when the MoCCM was set in a fiber optic system. An initial intensity change of the polarized light transmission, which dispersed perpendicular to the cell's attaching surface, was collected for 10 to 60 min, and then magnetic fields were applied parallel and perpendicular to the surface and light direction, respectively. The magnetic birefringence signals that originated from the magnetic orientation of intracellular molecules such as cytoskeletons apparently appeared when the magnetic fields were constant at 10 T. A statistical analysis with 15 experiments confirmed that the cellular components under 10 T magnetic fields caused a stronger alignment, which was transferred into polarizing light intensity that increased more than the case before exposure. Cellular conditions such as generation and cell density affected the magnetic birefringence signals.

  9. Intracellular activity of clinical concentrations of phenothiazines including thioridiazine against phagocytosed Staphylococcus aureus.

    PubMed

    Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Arroz, Maria Jorge; Amaral, Leonard

    2002-07-01

    The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3+ T cell production of IFN-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus. PMID:12127709

  10. Intracellular uptake and fluorescence imaging potential in tumor cell of zinc phthalocyanine.

    PubMed

    Avşar, Göktürk; Sari, Fatma Aslıhan; Yuzer, A Celil; Soylu, Hale Melis; Er, Ozge; Ince, Mine; Lambrecht, Fatma Yurt

    2016-05-30

    A near IR absorbing phthalocyanine bearing four binaphtyl group has been synthesized in order to investigate its cytotoxicity and intracellular uptake of sensitizer on MCF-7 (human breast cancer), MDAH (ovarian cancer), HeLa (human epitheloid cervix carcinoma), EMT-6 (mouse breast cancer) and WI-38 (human fibroblast lung) cell lines. ZnPc showed four time higher intracellular uptake in carcinoma cells (MCF-7) than normal (WI-38) cell lines. With the aim of studying in detail the biodistribution feature and tumor nuclear imaging capacity, ZnPc was also labeled with I-131. The efficiency of radiolabeled compound was 95±4.6%. In addition, ZnPc reveals to be very efficient singlet oxygen generators (ΦΔ=0.612 in DMSO) and promising PS for PDT application. In vitro fluorescence imaging study with MCF-7 cells showed that ZnPc localized in cytoplasm of the cells. This results showed that synthesized ZnPc is promising candidate for dual fluorescence/nuclear imaging breast cancer and shows potential PS for PDT application. PMID:27085647

  11. Location of sugars in multilamellar membranes at low hydration

    NASA Astrophysics Data System (ADS)

    Lenné, Thomas; Bryant, Gary; Garvey, Christopher J.; Keiderling, Uwe; Koster, Karen L.

    2006-11-01

    Severe dehydration is lethal for most biological species. However, there are a number of organisms which have evolved mechanisms to avoid damage during dehydration. One of these mechanisms is the accumulation of small solutes (e.g. sugars), which have been shown to preserve membranes by inhibiting deleterious phase changes at low hydration. Specifically, sugars reduce the gel to fluid phase transition temperatures of model lipid/water mixtures. However, there is a debate about the precise mechanism, the resolution of which hinges on the location of the sugars. In excess water, it has been observed using contrast variation SANS that the sugar concentration in the excess phase is higher than in the interlamellar region [Demé and Zemb, J. Appl. Crystallog. 33 (2000) 569]. This raises two questions regarding the location of the sugars at low hydrations: first, does the system phase separate to give a sugar/water phase in equilibrium with a lipid/water/sugar lamellar region (with different sugar concentrations); and second, is the sugar in the interlamellar region uniformly distributed, or does it concentrate preferentially either in close proximity to the lipids, or towards the center of the interbilayer region. In this paper we present the preliminary results of measurements using contrast variation SANS to determine the location of sugars in lipid/water mixtures.

  12. Leishmania mexicana mexicana: quantitative analysis of the intracellular cycle.

    PubMed

    Doyle, P S; Engel, J C; Gam, A A; Dvorak, J A

    1989-12-01

    The complete intracellular cycle of the Leishmania mexicana mexicana G. S. strain was quantified in human macrophages and in the mouse IC-21 macrophage line utilizing a culture system that allows the direct observation of individual intracellular parasites. A wide range of pre-replicative lag periods exists, implying that promastigotes may be in any phase of their DNA synthetic cycle when phagocytosed by the macrophage. Amastigotes replicated 2-3 times, after which the host cell died and liberated amastigotes that were taken up by other macrophages and continued to replicate. The mean amastigote population-doubling time in human macrophages (17.5 h) was not statistically different from promastigotes growing in axenic culture (16.4 h), but was nearly 2-fold less than amastigotes growing in mouse-derived IC-21 macrophages (33.7 h). These observations are markedly different from cover-glass culture assays of Leishmania-macrophage interactions and provide an unambiguous description of the intracellular cycle of Leishmania mexicana mexicana. PMID:2608309

  13. Role of envelope glycoproteins in intracellular virus maturation

    SciTech Connect

    Matsuoka, Y.

    1988-01-01

    The possible role viral glycoproteins in intracellular maturation was studied by using two different viruses, avian infectious bronchitis virus (IBV), a coronavirus, and Punta Toro virus (PTV), a bunyavirus. Using the antibiotic tunicamycin, which inhibits glycosylation of N-linked glycoproteins, it was shown that coronavirus particles are formed in the absence of glycosylation. Analysis of the protein composition of these particles indicated that they contain an unglycosylated form of the membrane-associated E1 glycoprotein but lack the E2 spike glycoprotein. A cDNA clone derived from the PTV M RNA genome segment, which encodes the G1 and G2 glycoproteins, was cloned into vaccinia virus. Studies by indirect immunofluorescence microscopy revealed that the glycoproteins synthesized from this recombinant were found to accumulate intracellularly at the Golgi complex, where virus budding usually takes place. Surface immunoprecipitation and {sup 125}I-protein A binding assays also demonstrated that a majority of the glycoproteins are retained intracellularly and are not transported to the cellular surface. The sequences which encode the G1 and G2 glycoproteins were independently cloned into vaccinia virus as well.

  14. Estimating the biophysical properties of neurons with intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Ye, Jingxin; Rozdeba, Paul J.; Morone, Uriel I.; Daou, Arij; Abarbanel, Henry D. I.

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V (t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  15. A mechanism of intracellular P2X receptor activation.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2012-08-17

    P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763

  16. Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial alteration.

    PubMed

    Debure, Laure; Vayssiere, Jean-Luc; Rincheval, Vincent; Loison, Fabien; Le Drean, Yves; Michel, Denis

    2003-08-01

    Clusterin is a puzzling protein upregulated in many diseased tissues, presented as either a survival or a death protein. The role of clusterin might depend on the final maturation and localization of the protein, which can be secreted or reside inside cells, either after in situ synthesis or uptake of extracellular clusterin. We studied the biological effects of intracellular clusterin and observed that clusterin forms containing the alpha-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2, and caspase inhibition was capable of rescuing cells expressing clusterin, which presented an altered mitochondrial permeability. We propose that, although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment. This activity might reconcile the opposite purported influences of clusterin on cell survival and explain how clusterin can be causally involved in neurodegeneration. PMID:12799419

  17. Forced Resurgence and Targeting of Intracellular Uropathogenic Escherichia coli Reservoirs

    PubMed Central

    Blango, Matthew G.; Ott, Elizabeth M.; Erman, Andreja; Veranic, Peter; Mulvey, Matthew A.

    2014-01-01

    Intracellular quiescent reservoirs of uropathogenic Escherichia coli (UPEC), which can seed the bladder mucosa during the acute phase of a urinary tract infection (UTI), are protected from antibiotic treatments and are extremely difficult to eliminate. These reservoirs are a potential source for recurrent UTIs that affect millions annually. Here, using murine infection models and the bladder cell exfoliant chitosan, we demonstrate that intracellular UPEC populations shift within the stratified layers of the urothelium during the course of a UTI. Following invasion of the terminally differentiated superficial layer of epithelial cells that line the bladder lumen, UPEC can multiply and disseminate, eventually establishing reservoirs within underlying immature host cells. If given access, UPEC can invade the superficial and immature bladder cells equally well. As infected immature host cells differentiate and migrate towards the apical surface of the bladder, UPEC can reinitiate growth and discharge into the bladder lumen. By inducing the exfoliation of the superficial layers of the urothelium, chitosan stimulates rapid regenerative processes and the reactivation and efflux of quiescent intracellular UPEC reservoirs. When combined with antibiotics, chitosan treatment significantly reduces bacterial loads within the bladder and may therefore be of therapeutic value to individuals with chronic, recurrent UTIs. PMID:24667805

  18. Probing cytoskeleton dynamics by intracellular particle transport analysis

    NASA Astrophysics Data System (ADS)

    Götz, M.; Hodeck, K. F.; Witzel, P.; Nandi, A.; Lindner, B.; Heinrich, D.

    2015-07-01

    All cellular functions arise from the transport of molecules through a heterogeneous, highly dynamic cell interior for intracellular signaling. Here, the impact of intracellular architecture and cytoskeleton dynamics on transport processes is revealed by high-resolution single particle tracking within living cells, in combination with time-resolved local mean squared displacement (I-MSD) analysis. We apply the I-MSD analysis to trajectories of 200 nm silica particles within living cells of Dictyostelium discoideum obtained by high resolution spinning disc confocal microscopy with a frame rate of 100 fps and imaging in one fixed focal plane. We investigate phases of motor-driven active transport and subdiffusion, normal diffusion, as well as superdiffusion with high spatial and temporal resolution. Active directed intracellular motion is attributed to microtubule associated molecular motor driven transport with average absolute velocities of 2.8 μm s-1 for 200 nm diameter particles. Diffusion processes of these particles within wild-type cells are found to exhibit diffusion constants ranging across two orders of magnitude from subdiffusive to superdiffusive behavior. This type of analysis might prove of ample importance for medical applications, like targeted drug treatment of cells by nano-sized carriers or innovative diagnostic assays.

  19. Intracellular Delivery System for Antibody–Peptide Drug Conjugates

    PubMed Central

    Berguig, Geoffrey Y; Convertine, Anthony J; Frayo, Shani; Kern, Hanna B; Procko, Erik; Roy, Debashish; Srinivasan, Selvi; Margineantu, Daciana H; Booth, Garrett; Palanca-Wessels, Maria Corinna; Baker, David; Hockenbery, David; Press, Oliver W; Stayton, Patrick S

    2015-01-01

    Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody–drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody–drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines. PMID:25669432

  20. Acoustically Propelled Nanomotors for Intracellular siRNA Delivery.

    PubMed

    Esteban-Fernández de Ávila, Berta; Angell, Chava; Soto, Fernando; Lopez-Ramirez, Miguel Angel; Báez, Daniela F; Xie, Sibai; Wang, Joseph; Chen, Yi

    2016-05-24

    An effective intracellular gene silencing strategy based on acoustically propelled nanowires modified with an interfering RNA's (siRNA) payload is described. The gold nanowires (AuNW) are wrapped with a Rolling Circle Amplification (RCA) DNA strand, which serves to anchor the siRNA therapy. The ultrasound (US)-powered propulsion of the AuNW leads to fast internalization and rapid intracellular movement and hence to an accelerated siRNA delivery and silencing response. To optimize the micromotor gene silencing procedure, the influence of motion, time, and siRNA dosage was investigated, leading up to a 94% silencing after few minutes treatment with US-propelled siRNA-AuNWs, and to a dramatic (∼13-fold) improvement in the silencing response compared to the static modified nanowires. The ability of the nanomotor-based method for gene silencing has been demonstrated by measuring the GFP silencing response in two different cell lines (HEK-293 and MCF-7) and using detailed control experiments. The viability of the cells after the nanomotors treatment was examined using the MCF-7 cancer cell line. The use of DNA structures carried by the US-propelled nanomotors for gene silencing represents an efficient tool that addresses the challenges associated with RNA transportation and intracellular delivery. Future implementation of nanomachines in gene therapy applications can be expanded into a co-delivery platform for therapeutics. PMID:27022755

  1. Twenty years of fluorescence imaging of intracellular chloride

    PubMed Central

    Arosio, Daniele; Ratto, Gian Michele

    2014-01-01

    Chloride homeostasis has a pivotal role in controlling neuronal excitability in the adult brain and during development. The intracellular concentration of chloride is regulated by the dynamic equilibrium between passive fluxes through membrane conductances and the active transport mediated by importers and exporters. In cortical neurons, chloride fluxes are coupled to network activity by the opening of the ionotropic GABAA receptors that provides a direct link between the activity of interneurons and chloride fluxes. These molecular mechanisms are not evenly distributed and regulated over the neuron surface and this fact can lead to a compartmentalized control of the intracellular concentration of chloride. The inhibitory drive provided by the activity of the GABAA receptors depends on the direction and strength of the associated currents, which are ultimately dictated by the gradient of chloride, the main charge carrier flowing through the GABAA channel. Thus, the intracellular distribution of chloride determines the local strength of ionotropic inhibition and influences the interaction between converging excitation and inhibition. The importance of chloride regulation is also underlined by its involvement in several brain pathologies, including epilepsy and disorders of the autistic spectra. The full comprehension of the physiological meaning of GABAergic activity on neurons requires the measurement of the spatiotemporal dynamics of chloride fluxes across the membrane. Nowadays, there are several available tools for the task, and both synthetic and genetically encoded indicators have been successfully used for chloride imaging. Here, we will review the available sensors analyzing their properties and outlining desirable future developments. PMID:25221475

  2. Quantification of intracellular payload release from polymersome nanoparticles

    PubMed Central

    Scarpa, Edoardo; Bailey, Joanne L.; Janeczek, Agnieszka A.; Stumpf, Patrick S.; Johnston, Alexander H.; Oreffo, Richard O. C.; Woo, Yin L.; Cheong, Ying C.; Evans, Nicholas D.; Newman, Tracey A.

    2016-01-01

    Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology. PMID:27404770

  3. Fatty Acid Signaling: The New Function of Intracellular Lipases

    PubMed Central

    Papackova, Zuzana; Cahova, Monika

    2015-01-01

    Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed. PMID:25674855

  4. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

    SciTech Connect

    Derian, C.K.; Friedman, P.A.

    1988-04-01

    The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

  5. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  6. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  7. Effects of Mutational Loss of Specific Intracellular Proteases on the Sporulation of Bacillus subtilis

    PubMed Central

    Hageman, James H.; Carlton, Bruce C.

    1973-01-01

    Two protease-deficient mutants of Bacillus subtilis 168 (Trp−) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring “neutral” protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover. Images PMID:4196247

  8. KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

    PubMed Central

    Goitre, Luca; Balzac, Fiorella; Degani, Simona; Degan, Paolo; Marchi, Saverio; Pinton, Paolo; Retta, Saverio Francesco

    2010-01-01

    KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the

  9. Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

    PubMed Central

    LeBlanc, Isabelle; Blot, Vincent; Bouchaert, Isabelle; Salamero, Jean; Goud, Bruno; Rosenberg, Arielle R.; Dokhélar, Marie-Christine

    2002-01-01

    Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system. PMID:11752179

  10. Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

    PubMed

    Wang, Tieyan; Chen, Qixian; Lu, Hongguang; Li, Wei; Li, Zaifen; Ma, Jianbiao; Gao, Hui

    2016-08-17

    The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression. PMID:27453033

  11. Intracellular collagen fibrils: evidence of an intracellular source from experiments with tendon fibroblasts and fibroblastic tumour cells.

    PubMed Central

    Michna, H

    1988-01-01

    This study was designed to substantiate one or both of the two hypotheses for the explanation of intracellular collagen fibrils in collagen-producing cells. The more obvious is the phagocytosis of extracellular collagen fibrils by the cell and the other is a form of autophagocytosis of newly synthesised collagenous products. Information was collected on fibroblasts from murine tendons after exercise and simultaneously stimulating collagen synthesis by treatment with an anabolic steroid hormone. Moreover, in vivo and in vitro fibroblastic tumour cells which demonstrate enhanced protein synthesis were also treated with the anabolic steroid. The findings of intracellular collagen fibrils in tendon fibroblasts and the sarcoma cells after experimentally stimulating collagen synthesis are discussed in the light of the hypothesis that the findings may represent steps of autophagocytosis of newly synthesised collagenous products in the absence of a control mechanism to remove collagenous products which cannot be secreted. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:3225213

  12. Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

    PubMed Central

    Kirby, Brett S.; Hanna, Gabi; Hendargo, Hansford C.

    2014-01-01

    Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo. PMID:25305182

  13. 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

  14. Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

    PubMed Central

    Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

    2007-01-01

    Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

  15. The PHR proteins: intracellular signaling hubs in neuronal development and axon degeneration.

    PubMed

    Grill, Brock; Murphey, Rodney K; Borgen, Melissa A

    2016-01-01

    During development, a coordinated and integrated series of events must be accomplished in order to generate functional neural circuits. Axons must navigate toward target cells, build synaptic connections, and terminate outgrowth. The PHR proteins (consisting of mammalian Phr1/MYCBP2, Drosophila Highwire and C. elegans RPM-1) function in each of these events in development. Here, we review PHR function across species, as well as the myriad of signaling pathways PHR proteins regulate. These findings collectively suggest that the PHR proteins are intracellular signaling hubs, a concept we explore in depth. Consistent with prominent developmental functions, genetic links have begun to emerge between PHR signaling networks and neurodevelopmental disorders, such as autism, schizophrenia and intellectual disability. Finally, we discuss the recent and important finding that PHR proteins regulate axon degeneration, which has further heightened interest in this fascinating group of molecules. PMID:27008623

  16. Intracellular Dual Fluorescent Lightup Bioprobes for Image-Guided Photodynamic Cancer Therapy.

    PubMed

    Han, Haijie; Jin, Qiao; Wang, Haibo; Teng, Wenzhuo; Wu, Jina; Tong, Hongxin; Chen, Tingting; Ji, Jian

    2016-07-01

    An intracellular dual fluorescent light-up bioprobe with aggregation-induced emission features and endogenously producing photosensitizer protoporphyrin IX (PpIX) abilities is designed and synthesized. The bioprobe is nonemissive in physiological environment. However, the bioprobe can selectively light up cancer cells with blue fluorescence of tetraphenylene (TPE) and red fluorescence of PpIX, owing to the release of TPE and methyl aminolevulinate after targeted internalization by cancer cells. Moreover, upon endogenous generation and accumulation of PpIX in cancer cells, efficient photodynamic ablation of cancer cells after light irradiation is demonstrated with easy regulation for optimal therapeutic efficacy. The design of such dual fluorescent light-up bioprobes might provide a new opportunity for targeted and image-guided photodynamic cancer therapy. PMID:27322139

  17. Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking.

    PubMed

    Schubert, Marcel; Steude, Anja; Liehm, Philipp; Kronenberg, Nils M; Karl, Markus; Campbell, Elaine C; Powis, Simon J; Gather, Malte C

    2015-08-12

    We report on a laser that is fully embedded within a single live cell. By harnessing natural endocytosis of the cell, we introduce a fluorescent whispering gallery mode (WGM) microresonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells. Our approach can be applied to different cell types, and cells with microresonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing, and adaptive imaging. PMID:26186167

  18. Intracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

    PubMed Central

    Decleva, Eva; Menegazzi, Renzo; Fasolo, Alba; Defendi, Federica; Sebastianutto, Michele; Dri, Pietro

    2013-01-01

    Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn2+, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2− and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn2+ results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2− generated within gp91phox in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux. PMID:23578765

  19. Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.

    PubMed

    Smyllie, Nicola J; Pilorz, Violetta; Boyd, James; Meng, Qing-Jun; Saer, Ben; Chesham, Johanna E; Maywood, Elizabeth S; Krogager, Toke P; Spiller, David G; Boot-Handford, Raymond; White, Michael R H; Hastings, Michael H; Loudon, Andrew S I

    2016-07-25

    Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation [1, 2]. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation [3, 4]. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension [5-7]. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock. PMID:27374340

  20. Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Tseng, Peter

    Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to

  1. Surface functionalized mesoporous silica nanoparticles for intracellular drug delivery

    NASA Astrophysics Data System (ADS)

    Vivero-Escoto, Juan Luis

    Mesoporous silica nanoparticles (MSNs) are a highly promising platform for intracellular controlled release of drugs and biomolecules. Despite that the application of MSNs in the field of intracellular drug delivery is still at its infancy very exciting breakthroughs have been achieved in the last years. A general review of the most recent progress in this area of research is presented, including a description of the latest findings on the pathways of entry into live mammalian cells together with the intracellular trafficking, a summary on the contribution of MSNs to the development of site-specific drug delivery systems, a report on the biocompatibility of this material in vitro andin vivo, and a discussion on the most recent breakthroughs in the synthesis and application of stimuli-responsive mesoporous silica-based delivery vehicles. A gold nanoparticles (AuNPs)-capped MSNs-based intracellular photoinduced drug delivery system (PR-AuNPs-MSNs) for the controlled release of anticancer drug inside of human fibroblast and liver cells was synthesized and characterized. We found that the mesoporous channels of MSNs could be efficiently capped by the photoresponsive AuNPs without leaking the toxic drug, paclitaxel, inside of human cells. Furthermore, we demonstrated that the cargo-release property of this PR-AuNPs-MSNs system could be easily photo-controlled under mild and biocompatible conditions in vitro. In collaboration with Renato Mortera (a visiting student from Italy), a MSNs based intracellular delivery system for controlled release of cell membrane impermeable cysteine was developed. A large amount of cysteine molecules were covalently attached to the silica surface of MSNs through cleavable disulfide linkers. These cysteine-containing nanoparticles were efficiently endocytosed by human cervical cancer cells HeLa. These materials exhibit 450 times higher cell growth inhibition capability than that of the conventional N-acetylcysteine prodrug. The ability to

  2. Intracellular calcium dynamics dependent on defined microtopographical features of titanium.

    PubMed

    Staehlke, Susanne; Koertge, Andreas; Nebe, Barbara

    2015-04-01

    Detailed insights into the complex cellular behavior at the biomaterial interface are crucial for the improvement of implant surfaces with respect to their acceptance and integration. The cells perceive microtopographical features and, in consequence, rearrange their adhesion structures like the actin cytoskeleton and adaptor proteins. But little is known about whether these altered cellular phenotypes have consequences for intracellular calcium signaling and its dynamics. To elucidate if an artificial, geometrical microtopography influences calcium ion (Ca(2+)) mobilization in osteoblasts, human MG-63 cells were stained with the calcium dye Fluo 3-acetoxymethyl ester and set on defined silicon-titanium (Ti) arrays with regular pillar structures (P5, 5 × 5 × 5 μm) and compared with planar Ti. To induce an immediate calcium signal, cells were stimulated with adenosine 5'-triphosphate (ATP). Interestingly, osteoblasts on micropillars expressing a shortened actin cytoskeleton were hampered in their calcium mobilization potential in signal height as well duration. Even the basal level of the intracellular Ca(2+) concentration was reduced, which was accompanied by a disturbed fibronectin synthesis. The expression of the voltage-sensitive calcium channels Cav1.2, Cav1.3 (L-type) and Cav3.1, Cav3.2, Cav3.3 (T-type) as well as the signaling proteins phospho-AKT and phospho-GSK3α/β remained unaffected on pillars. The topography-dependent calcium dynamics observed here provide new insights into how topographical cues alter cell functions - via the intracellular Ca(2+) signaling. PMID:25678115

  3. Legionella pneumophila requires polyamines for optimal intracellular growth.

    PubMed

    Nasrallah, Gheyath K; Riveroll, Angela L; Chong, Audrey; Murray, Lois E; Lewis, P Jeffrey; Garduño, Rafael A

    2011-09-01

    The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  4. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    PubMed Central

    Veetil, Jithesh V.; Jin, Sha; Ye, Kaiming

    2012-01-01

    Background One of the major hurdles in studying diabetes pathophysiology is the lack of adequate methodology that allows for direct and real-time determination of glucose transport and metabolism in cells and tissues. In this article, we present a new methodology that adopts frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) to visualize and quantify the dynamics of intracellular glucose within living cells using a biosensor protein based on fluorescence resonance energy transfer (FRET). Method The biosensor protein was developed by fusing a FRET pair, an AcGFP1 donor and a mCherry acceptor to N- and C- termini of a mutant glucose-binding protein (GBP), respectively. The probe was expressed and biosynthesized inside the cells, offering continuous monitoring of glucose dynamics in real time through fluorescence lifetime imaging microscopy (FLIM) measurement. Results We transfected the deoxyribonucleic acid of the AcGFP1-GBP-mCherry sensor into murine myoblast cells, C2C12, and continuously monitored the changes in intracellular glucose concentrations in response to the variation in extracellular glucose, from which we determined glucose uptake and clearance rates. The distribution of intracellular glucose concentration was also characterized. We detected a high glucose concentration in a region close to the cell membrane and a low glucose concentration in a region close to the nucleus. The monoexponential decay of AcGFP1 was distinguished using FD-FLIM. Conclusions This work enables continuous glucose monitoring (CGM) within living cells using FD-FLIM and a biosensor protein. The sensor protein developed offers a new means for quantitatively analyzing glucose homeostasis at the cellular level. Data accumulated from these studies will help increase our understanding of the pathology of diabetes. PMID:23294772

  5. Fluorescence detection of intracellular cadmium with Leadmium Green.

    PubMed

    Malaiyandi, Latha M; Sharthiya, Harsh; Dineley, Kirk E

    2016-08-01

    Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd(2+)) and lead (Pb(2+)). Leadmium Green has been used in various paradigms, such as tracking Cd(2+) sequestration in plant cells, heavy metal export in protozoa, and Pb(2+) absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd(2+), Pb(2+), and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer's claims, Leadmium Green was sensitive to Cd(2+) (KD ~600 nM) and also Pb(2+) (KD ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca(2+), Co(2+), Mn(2+) and Ni(2+). Leadmium Green also responded to Zn(2+) with a KD of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd(2+) or Zn(2+) in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn(2+) was much less than that produced by Cd(2+). When tested in a model of oxidant-induced liberation of endogenous Zn(2+), Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd(2+), particularly in models where Cd(2+) accumulates rapidly, and when concomitant fluctuations of intracellular Zn(2+) are minimal. PMID:27260023

  6. Intracellular Mono-ADP-Ribosylation in Signaling and Disease

    PubMed Central

    Bütepage, Mareike; Eckei, Laura; Verheugd, Patricia; Lüscher, Bernhard

    2015-01-01

    A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD+)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases. PMID:26426055

  7. Transient fluctuations of intracellular zinc ions in cell proliferation

    SciTech Connect

    Li, Yuan; Maret, Wolfgang

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  8. Diagnostic Accuracy of Intracellular Mycobacterium tuberculosis Detection for Tuberculous Meningitis

    PubMed Central

    Feng, Guo-dong; Shi, Ming; Ma, Lei; Chen, Ping; Wang, Bing-ju; Zhang, Min; Chang, Xiao-lin; Su, Xiu-chu; Yang, Yi-ning; Fan, Xin-hong; Dai, Wen; Liu, Ting-ting; He, Ying; Bian, Ting; Duan, Li-xin; Li, Jin-ge; Hao, Xiao-ke; Liu, Jia-yun; Xue, Xin; Song, Yun-zhang; Wu, Hai-qin; Niu, Guo-qiang; Zhang, Li; Han, Cui-juan; Lin, Hong; Lin, Zhi-hui; Liu, Jian-jun; Jian, Qian; Zhang, Jin-she; Tian, Ye; Zhou, Bai-yu; Wang, Jing; Xue, Chang-hu; Han, Xiao-fang; Wang, Jian-feng; Wang, Shou-lian

    2014-01-01

    Rationale: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity. Objectives: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis. Methods: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6–expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients’ final diagnosis. Measurements and Main Results: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6–6.7%), compared with 82.9% (95% confidence interval, 77.4–87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8–80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4–93.8%) and 90.0% (95% confidence interval, 75.4–96.7%), respectively. Conclusions: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis. PMID:24450377

  9. Quenching methods for the analysis of intracellular metabolites.

    PubMed

    Wahrheit, Judith; Heinzle, Elmar

    2014-01-01

    Sampling and quenching methods for intracellular metabolite analysis in mammalian cells in adherent and suspension culture are described. Quenching of adherent cells is achieved by application of hot air after removal of the supernatant by suction. For suspension cultures, the addition of excess ice-cold saline results in a rapid inactivation of metabolism and significant dilution of extracellular metabolites. Medium carryover is prevented by rinsing the cells with washing solution. Separation of supernatant from suspension cells via centrifugation is incomplete due to required short centrifugation time. Thus, it is necessary to determine the reproducible cell recovery after quenching. PMID:24297418

  10. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  11. Intracellular iron minerals in a dissimilatory iron-reducing bacterium.

    PubMed

    Glasauer, Susan; Langley, Sean; Beveridge, Terry J

    2002-01-01

    Among prokaryotes, there are few examples of controlled mineral formation; the formation of crystalline iron oxides and sulfides [magnetite (Fe3O4) or greigite (Fe3S4)] by magnetotactic bacteria is an exception. Shewanella putrefaciens CN32, a Gram-negative, facultative anaerobic bacterium that is capable of dissimilatory iron reduction, produced microscopic intracellular grains of iron oxide minerals during growth on two-line ferrihydrite in a hydrogen-argon atmosphere. The minerals, formed at iron concentrations found in the soil and sedimentary environments where these bacteria are active, could represent an unexplored pathway for the cycling of iron by bacteria. PMID:11778045

  12. Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

    PubMed Central

    Pakula, A A; Young, V B; Sauer, R T

    1986-01-01

    Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type. Images PMID:2947238

  13. Intracellular coagulation inhibits the extraction of proteins from Prochloron

    NASA Technical Reports Server (NTRS)

    Fall, R.; Lewin, R. A.; Fall, L. R.

    1983-01-01

    Protein extraction from the prokaryotic alga Prochloron LP (isolated from the ascidian host Lissoclinum patella) was complicated by an irreversible loss of cell fragility in the isolated algae. Accompanying this phenomenon, which is termed intracellular coagulation, was a redistribution of thylakoids around the cell periphery, a loss of photosynthetic O2 production, and a drastic decrease in the extractability of cell proteins. Procedures are described for the successful preparation and transport of cell extracts yielding the enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as other soluble proteins.

  14. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    PubMed

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well. PMID:24482125

  15. Metabolic Interdependence of Obligate Intracellular Bacteria and Their Insect Hosts†

    PubMed Central

    Zientz, Evelyn; Dandekar, Thomas; Gross, Roy

    2004-01-01

    Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism—that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts. PMID:15590782

  16. Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.

    PubMed

    Zielonka, Jacek; Kalyanaraman, B

    2010-04-15

    Hydroethidine (HE; or dihydroethidium) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E(+)). In biological systems, another red fluorescent product, ethidium, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review the methods to selectively detect the superoxide-specific product (2-OH-E(+)) and the factors affecting its levels in cellular and biological systems. The most important conclusion of this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E(+), to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX red) with superoxide is similar to the reactivity of HE with superoxide, and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or MitoSOX) as well. PMID:20116425

  17. An atmospheric-pressure cold plasma leads to apoptosis in Saccharomyces cerevisiae by accumulating intracellular reactive oxygen species and calcium

    NASA Astrophysics Data System (ADS)

    Ma, R. N.; Feng, H. Q.; Liang, Y. D.; Zhang, Q.; Tian, Y.; Su, B.; Zhang, J.; Fang, J.

    2013-07-01

    A non-thermal plasma is known to induce apoptosis of various cells but the mechanism is not yet clear. A eukaryotic model organism Saccharomyces cerevisiaewas used to investigate the cellular and biochemical regulations of cell apoptosis and cell cycle after an atmospheric-pressure cold plasma treatment. More importantly, intracellular calcium (Ca2+) was first involved in monitoring the process of plasma-induced apoptosis in this study. We analysed the cell apoptosis and cell cycle by flow cytometry and observed the changes in intracellular reactive oxygen species (ROS) and Ca2+ concentration, cell mitochondrial membrane potential (Δψm) as well as nuclear DNA morphology via fluorescence staining assay. All experimental results indicated that plasma-generated ROS leads to the accumulation of intracellular ROS and Ca2+ that ultimately contribute to apoptosis associated with cell cycle arrest at G1 phase through depolarization of Δψm and fragmenting nuclear DNA. This work provides a novel insight into the physical and biological mechanism of apoptosis induced by a plasma which could benefit for promoting the development of plasmas applied to cancer therapy.

  18. Quantitative modelling of the effects of selected intracellular metabolites on pH in fish white muscle

    PubMed

    Arthur; West; Hochachka

    1997-01-01

    A model is presented that provides guidelines to the identification of key experimental variables influencing proton balance and intracellular pH in vertebrate white muscle. We have drawn on data from the literature on rainbow trout (Oncorhynchus mykiss) in an attempt to quantify the influence of metabolic, ionic and transport components of proton generation and proton consumption after exercise. Only minor changes in proton balance and in calculated intracellular pH were caused by considering changes in the concentration of bicarbonate or including the acid­base characteristics of purine nucleotides. Intracellular pH, as estimated by the model, was more acidic at some time points in recovery compared with in vivo measurements, and this would appear to result mainly from inaccuracies in quantifying the phosphate component of proton buffering. Nevertheless, the model was able to simulate the typical pattern of muscle acidosis and recovery observed for trout, including the transient post-exercise acidification and the slow recovery rate. As with previous pHi models, comparison of model estimates with experimental observations is essential in this approach in order to identify whether all of the relevant metabolic processes have been considered for accurate quantification of proton balance within the white muscle compartment. PMID:9319037

  19. Signaling Microdomains Regulate Inositol 1,4,5-Trisphosphate-Mediated Intracellular Calcium Transients in Cultured Neurons

    PubMed Central

    Jacob, Simon N.; Choe, Chi-Un; Uhlen, Per; DeGray, Brenda; Yeckel, Mark F.; Ehrlich, Barbara E.

    2010-01-01

    Ca2+signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, we correlated the subcellular distribution of components of the InsP3 pathway with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKIγ) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKIγ or disrupting the CGB–InsP3R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca2+signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+signaling in different types of neurons. PMID:15772345

  20. FRET-Based Nanobiosensors for Imaging Intracellular Ca²⁺ and H⁺ Microdomains.

    PubMed

    Zamaleeva, Alsu I; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne

    2015-01-01

    Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca(2+) and pH transients using live-cell fluorescence imaging. PMID:26404317

  1. Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

    PubMed

    Ali, F; Khan, A Q; Khan, R; Sultana, S

    2016-02-01

    Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar. PMID:25855085

  2. Hydroethidine- and Mito-SOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth

    PubMed Central

    Zielonka, Jacek; Kalyanaraman, B.

    2010-01-01

    Hydroethidine (or dihydroethidium) (HE) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E+). In biological systems, another red fluorescent product, ethidium (E+), is also formed, usually at a much higher concentration than 2-OH-E+. In this article, we have reviewed the methods to selectively detect the superoxide-specific product (2-OH-E+) and the factors affecting its levels in cellular and biological systems. The most important conclusion of the present review is that it is nearly impossible to assess the intracellular levels of the superoxide specific product, 2-OH-E+, using the confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E+, in order to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX Red ®) with superoxide is similar to the reactivity of HE with superoxide and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or Mito-SOX) as well. PMID:20116425

  3. Regulation of B Cell Differentiation by Intracellular Membrane-Associated Proteins and microRNAs: Role in the Antibody Response.

    PubMed

    Lou, Zheng; Casali, Paolo; Xu, Zhenming

    2015-01-01

    B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes, and autophagosomes) and protein factors specifically associated with these membranes, including Rab7, Atg5, and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, class switch DNA recombination (CSR)/somatic hypermutation (SHM), and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation, and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulating AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses. PMID:26579118

  4. Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

    PubMed

    Kadiri, Lolahon R; Kwan, Alex C; Webb, Watt W; Harris-Warrick, Ronald M

    2011-09-01

    Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron. PMID:21676929

  5. Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

    PubMed

    Wu, Li-Hua; Li, Peng; Zhao, Qing-Li; Piao, Jin-Lan; Jiao, Yu-Fei; Kadowaki, Makoto; Kondo, Takashi

    2014-11-01

    Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-β-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway. PMID:25187044

  6. Hybrid micro-/nanogels for optical sensing and intracellular imaging

    PubMed Central

    Wu, Weitai; Zhou, Shuiqin

    2010-01-01

    Hybrid micro-/nanogels are playing an increasing important part in a diverse range of applications, due to their tunable dimensions, large surface area, stable interior network structure, and a very short response time. We review recent advances and challenges in the developments of hybrid micro-/nanogels toward applications for optical sensing of pH, temperature, glucose, ions, and other species as well as for intracellular imaging. Due to their unique advantages, hybrid micro-/nanogels as optical probes are attracting substantial interests for continuous monitoring of chemical parameters in complex samples such as blood and bioreactor fluids, in chemical research and industry, and in food quality control. In particular, their intracellular probing ability enables the monitoring of the biochemistry and biophysics of live cells over time and space, thus contributing to the explanation of intricate biological processes and the development of novel diagnoses. Unlike most other probes, hybrid micro-/nanogels could also combine other multiple functions into a single probe. The rational design of hybrid micro-/nanogels will not only improve the probing applications as desirable, but also implement their applications in new arenas. With ongoing rapid advances in bionanotechnology, the well-designed hybrid micro-/nanogel probes will be able to provide simultaneous sensing, imaging diagnosis, and therapy toward clinical applications. PMID:22110866

  7. In Vivo Detection of Intracellular Signaling Pathways in Developing Thymocytes

    PubMed Central

    Zúñiga-Pflücker, Juan Carlos

    2000-01-01

    Information regarding the intracellular signaling processes that occur during the development of T cells has largely been obtained with the use of transgenic mouse models, which although providing invaluable information are time consuming and costly. To this end, we have developed a novel system that facilitates the In Vivo analysis of signal transduction pathways during T-lymphocyte development. This approach uses reporter-plasmids for the detection of intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent protein kinase. Reporter-plasmids are transfected into thymocytes in fetal thymic organ culture by accelerated DNA/particle bombardment (gene gun), and the activation of a signaling pathway is determined in the form of a standard luciferase assay. Importantly, this powerful technique preserves the structural integrity of the thymus, and will provide an invaluable tool to study how thymocytes respond to normal environmental stimuli encountered during differentiation within the thymic milieu. Thus, this method allows for the monitoring of signals that occur in a biological time frame, such as during differentiation, and within the natural environment of differentiating cells. PMID:11293810

  8. Cell fate reprogramming by control of intracellular network dynamics

    NASA Astrophysics Data System (ADS)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  9. Intracellular BK(Ca) (iBK(Ca)) channels.

    PubMed

    Singh, Harpreet; Stefani, Enrico; Toro, Ligia

    2012-12-01

    The large conductance calcium- and voltage-activated potassium channel (BK(Ca)) is widely expressed at the plasma membrane. This channel is involved in a variety of fundamental cellular functions including excitability, smooth muscle contractility, and Ca(2+) homeostasis, as well as in pathological situations like proinflammatory responses in rheumatoid arthritis, and cancer cell proliferation. Immunochemical, biochemical and pharmacological studies from over a decade have intermittently shown the presence of BK(Ca) in intracellular organelles. To date, intracellular BK(Ca) (iBK(Ca)) has been localized in the mitochondria, endoplasmic reticulum, nucleus and Golgi apparatus but its functional role remains largely unknown except for the mitochondrial BK(Ca) whose opening is thought to play a role in protecting the heart from ischaemic injury. In the nucleus, pharmacology suggests a role in regulating nuclear Ca(2+), membrane potential and eNOS expression. Establishing the molecular correlates of iBK(Ca), the mechanisms defining iBK(Ca) organelle-specific targeting, and their modulation are challenging questions. This review summarizes iBK(Ca) channels, their possible functions, and efforts to identify their molecular correlates. PMID:22930268

  10. Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters.

    PubMed

    Van Laer, Koen; Dick, Tobias P

    2016-01-31

    It is increasingly apparent that nature evolved peroxiredoxins not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Here we ask whether the H2O2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H2O2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H2O2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H2O2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H2O2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H2O2 homeostasis and Prx signaling. PMID:26810074

  11. Monitoring the intracellular calcium response to a dynamic hypertonic environment

    PubMed Central

    Huang, Xiaowen; Yue, Wanqing; Liu, Dandan; Yue, Jianbo; Li, Jiaqian; Sun, Dong; Yang, Mengsu; Wang, Zuankai

    2016-01-01

    The profiling of physiological response of cells to external stimuli at the single cell level is of importance. Traditional approaches to study cell responses are often limited by ensemble measurement, which is challenging to reveal the complex single cell behaviors under a dynamic environment. Here we report the development of a simple microfluidic device to investigate intracellular calcium response to dynamic hypertonic conditions at the single cell level in real-time. Interestingly, a dramatic elevation in the intracellular calcium signaling is found in both suspension cells (human leukemic cell line, HL-60) and adherent cells (lung cancer cell line, A549), which is ascribed to the exposure of cells to the hydrodynamic stress. We also demonstrate that the calcium response exhibits distinct single cell heterogeneity as well as cell-type-dependent responses to the same stimuli. Our study opens up a new tool for tracking cellular activity at the single cell level in real time for high throughput drug screening. PMID:27004604

  12. Intracellular imaging of Qdots-labeled DNA in cyanobacteria.

    PubMed

    Loukanov, Alexandre; Zhelyazkov, Veselin; Hihara, Yukako; Nakabayashi, Seiichiro

    2016-05-01

    In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots-streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots-DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots-DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long-term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots-labeled plasmid DNA was utilized for the first time for long-term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots-labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447-452, 2016. © 2016 Wiley Periodicals, Inc. PMID:26957226

  13. Multistability and dynamic transitions of intracellular Min protein patterns.

    PubMed

    Wu, Fabai; Halatek, Jacob; Reiter, Matthias; Kingma, Enzo; Frey, Erwin; Dekker, Cees

    2016-01-01

    Cells owe their internal organization to self-organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction-diffusion patterns that pervade biology at many scales. PMID:27279643

  14. Fluorescent nanosensors for intracellular measurements: synthesis, characterization, calibration, and measurement

    PubMed Central

    Desai, Arpan S.; Chauhan, Veeren M.; Johnston, Angus P. R.; Esler, Tim; Aylott, Jonathan W.

    2013-01-01

    Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy. PMID:24474936

  15. Inhibitor of apoptosis proteins as intracellular signaling intermediates.

    PubMed

    Kocab, Andrew J; Duckett, Colin S

    2016-01-01

    Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates. PMID:26462035

  16. Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

    PubMed

    Yamochi, T; Hashimoto, S; Amo, A; Goto, H; Yamanaka, M; Inoue, M; Nakaoka, Y; Morimoto, Y

    2016-08-01

    Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes. PMID:26364763

  17. Charcot–Marie–Tooth disease and intracellular traffic

    PubMed Central

    Bucci, Cecilia; Bakke, Oddmund; Progida, Cinzia

    2012-01-01

    Mutations of genes whose primary function is the regulation of membrane traffic are increasingly being identified as the underlying causes of various important human disorders. Intriguingly, mutations in ubiquitously expressed membrane traffic genes often lead to cell type- or organ-specific disorders. This is particularly true for neuronal diseases, identifying the nervous system as the most sensitive tissue to alterations of membrane traffic. Charcot–Marie–Tooth (CMT) disease is one of the most common inherited peripheral neuropathies. It is also known as hereditary motor and sensory neuropathy (HMSN), which comprises a group of disorders specifically affecting peripheral nerves. This peripheral neuropathy, highly heterogeneous both clinically and genetically, is characterized by a slowly progressive degeneration of the muscle of the foot, lower leg, hand and forearm, accompanied by sensory loss in the toes, fingers and limbs. More than 30 genes have been identified as targets of mutations that cause CMT neuropathy. A number of these genes encode proteins directly or indirectly involved in the regulation of intracellular traffic. Indeed, the list of genes linked to CMT disease includes genes important for vesicle formation, phosphoinositide metabolism, lysosomal degradation, mitochondrial fission and fusion, and also genes encoding endosomal and cytoskeletal proteins. This review focuses on the link between intracellular transport and CMT disease, highlighting the molecular mechanisms that underlie the different forms of this peripheral neuropathy and discussing the pathophysiological impact of membrane transport genetic defects as well as possible future ways to counteract these defects. PMID:22465036

  18. Methods to follow intracellular trafficking of cell-penetrating peptides.

    PubMed

    Pärnaste, Ly; Arukuusk, Piret; Zagato, Elisa; Braeckmans, Kevin; Langel, Ülo

    2016-01-01

    Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems. PMID:26460120

  19. Structural rearrangement of the intracellular domains during AMPA receptor activation.

    PubMed

    Zachariassen, Linda G; Katchan, Ljudmila; Jensen, Anna G; Pickering, Darryl S; Plested, Andrew J R; Kristensen, Anders S

    2016-07-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  20. Extracellular and Intracellular Actions of Sphingosine-1-Phosphate

    PubMed Central

    Strub, Graham M.; Maceyka, Michael; Hait, Nitai C.; Milstien, Sheldon; Spiegel, Sarah

    2009-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator with crucial roles in a wide variety of cellular functions across a broad range of organisms. Though a simple molecule in structure, S1P functions are complex. The formation of S1P is catalyzed by one of two sphingosine kinases that have differential cellular distributions as well as both overlapping and opposing functions and which are activated by many different stimuli. S1P can act on a family of G protein-coupled receptors (S1PRs) that are also differentially expressed in different cell types, which influences the cellular responses to S1P. In addition to acting on receptors located on the plasma membrane, S1P can also function inside the cell, independently of S1PRs. It also appears that both the intracellular location and the isotype of sphingosine kinase involved are major determinants of inside-out signaling of S1P in response to many extracellular stimuli. This chapter is focused on the current literature on extracellular and intracellular actions of S1P PMID:20919652

  1. Imaging the intracellular degradation of biodegradable polymer nanoparticles

    PubMed Central

    Barthel, Anne-Kathrin; Dass, Martin; Dröge, Melanie; Cramer, Jens-Michael; Baumann, Daniela; Urban, Markus; Landfester, Katharina; Mailänder, Volker

    2014-01-01

    Summary In recent years, the development of smart drug delivery systems based on biodegradable polymeric nanoparticles has become of great interest. Drug-loaded nanoparticles can be introduced into the cell interior via endocytotic processes followed by the slow release of the drug due to degradation of the nanoparticle. In this work, poly(L-lactic acid) (PLLA) was chosen as the biodegradable polymer. Although common degradation of PLLA has been studied in various biological environments, intracellular degradation processes have been examined only to a very limited extent. PLLA nanoparticles with an average diameter of approximately 120 nm were decorated with magnetite nanocrystals and introduced into mesenchymal stem cells (MSCs). The release of the magnetite particles from the surface of the PLLA nanoparticles during the intracellular residence was monitored by transmission electron microscopy (TEM) over a period of 14 days. It was demonstrated by the release of the magnetite nanocrystals from the PLLA surface that the PLLA nanoparticles do in fact undergo degradation within the cell. Furthermore, even after 14 days of residence, the PLLA nanoparticles were found in the MSCs. Additionally, the ultrastructural TEM examinations yield insight into the long term intercellular fate of these nanoparticles. From the statistical analysis of ultrastructural details (e.g., number of detached magnetite crystals, and the number of nanoparticles in one endosome), we demonstrate the importance of TEM studies for such applications in addition to fluorescence studies (flow cytometry and confocal laser scanning microscopy). PMID:25383302

  2. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

  3. Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters

    PubMed Central

    Van Laer, Koen; Dick, Tobias P.

    2016-01-01

    It is increasingly apparent that nature evolved peroxiredoxins not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Here we ask whether the H2O2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H2O2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H2O2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H2O2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H2O2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H2O2 homeostasis and Prx signaling. PMID:26810074

  4. Intracellular inclusions of a n-alkane-grown Arthrobacter

    SciTech Connect

    Griffin, W.M.; Traxler, R.W.

    1983-01-01

    The ultrastructural changes associated with the growth of a marine Arthrobacter sp. on n-hexadecane were demonstrated by transmission electron microscopy. Multiple electron translucent areas (ETI), similar to inclusions described by other investigators, were found in hydrocarbon-grown cells but not in peptone-grown cells. Stereological planimetry demonstrated that the ETI occupy as much as 40% of the hydrocarbon-grown cell's volume. Electron dense structures (EDI) were observed in hexadecane and in peptone-grown cells. Gas chromatographic analysis indicated traces of n-hexadecane associated with the hydrocarbon-grown cells: however, the hydrocarbon levels were not high enough to suggest the presence of hydrocarbon inclusions in the cells. Solvent partition studies with disrupted UC-n-hexadecane-grown cells suggested that the intracellular radioactivity was associated with polar compounds. Acrylate-inhibited, hydrocarbon-grown Arthrobacter sp. did not form ETI, suggesting that the formation of ETI involves the participation of Coenzyme A. Thin-layer and gas chromatography indicated that the major intracellular components were glycerides, with one or more R groups composed of palmitic acid and free palmitic acid. 27 references, 5 figures, 2 tables.

  5. Intracellular cytokine production and cognition in healthy older adults.

    PubMed

    Simpson, Ellen E A; Hodkinson, Claire F; Maylor, Elizabeth A; McCormack, Jacqueline M; Rae, Gordon; Strain, Sean; Alexander, H Denis; Wallace, Julie M W

    2013-10-01

    Elevated concentrations of the pro-inflammatory cytokines IL-1β and IL-6 have been associated with impaired cognitive performance. There are, however, few studies that have examined the relationship between cytokine production and specific aspects of cognition in healthy older individuals. Two-colour flow cytometry was used to determine intracellular cytokine production by activated monocytes, and neuropsychological tests were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) in 93 apparently healthy men and women aged 55-70 years. A series of hierarchical regression analyses was carried out to examine the contribution of IL-1β and IL-6 (% expression and production (antibody binding capacity (ABC))) to recognition, attention and working memory, after controlling for socio-demographic variables (age, sex and social class). IL-1β% expression and IL-6 production predicted aspects of working memory. Recognition memory was found to be sensitive to the a