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Sample records for acidic intracellular compartments

  1. 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

  2. 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

  3. A polymer-Triton X-100 conjugate capable of PH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments.

    PubMed

    Duncan, R; Ferruti, P; Sgouras, D; Tuboku-Metzger, A; Ranucci, E; Bignotti, F

    1994-01-01

    Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml). PMID:7858959

  4. 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

  5. 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

  6. Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting.

    PubMed Central

    Perrin-Cocon, L A; Marche, P N; Villiers, C L

    1999-01-01

    In the present study, we describe a method to specifically isolate intracellular compartments containing endocytosed antigen. We have demonstrated that isolated compartments represent a small proportion of the intracellular material, highly enriched in antigen. Antigen-containing vesicles are specifically sorted from other intracellular compartments, such as endoplasmic reticulum or Golgi apparatus, and from the plasma membrane. They remain functional in vitro since they can be acidified, and the antigen inside has been found to be partially proteolysed. In macrophages, kinetic analysis has revealed that the antigen is first found in compartments of endosomal density, carrying Rab 5 and Rab 7, then in late compartments of lysosomal density, which are rich in proteases. The global protein content of the compartments was mapped by two-dimensional electrophoresis. In B lymphocytes, this method has allowed the isolation of endocytic compartments emerging from receptor-mediated endocytosis of the antigen. After 2 h of chase, the antigen reached vesicles containing large amounts of MHC-class II molecules, invariant chain and human leucocyte antigen-DM, where peptide loading can occur. PMID:9931307

  7. Chemoattractant-Regulated Mobilization of a Novel Intracellular Compartment in Human Neutrophils

    NASA Astrophysics Data System (ADS)

    Borregaard, N.; Miller, L. J.; Springer, T. A.

    1987-09-01

    A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revelaed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.

  8. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

    NASA Astrophysics Data System (ADS)

    Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

  9. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

    PubMed

    Huber, Matthias C; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials. PMID:25362355

  10. Amino Acid Sensing by mTORC1: Intracellular Transporters Mark the Spot.

    PubMed

    Goberdhan, Deborah C I; Wilson, Clive; Harris, Adrian L

    2016-04-12

    Cell metabolism and growth are matched to nutrient availability via the amino-acid-regulated mechanistic target of rapamycin complex 1 (mTORC1). Transporters have emerged as important amino acid sensors controlling mTOR recruitment and activation at the surface of multiple intracellular compartments. Classically, this has involved late endosomes and lysosomes, but now, in a recent twist, also the Golgi apparatus. Here we propose a model in which specific amino acids in assorted compartments activate different mTORC1 complexes, which may have distinct drug sensitivities and functions. We will discuss the implications of this for mTORC1 function in health and disease. PMID:27076075

  11. Self-assembled hydrogel fibers for sensing the multi-compartment intracellular milieu

    PubMed Central

    Vemula, Praveen Kumar; Kohler, Jonathan E.; Blass, Amy; Williams, Miguel; Xu, Chenjie; Chen, Lynna; Jadhav, Swapnil R.; John, George; Soybel, David I.; Karp, Jeffrey M.

    2014-01-01

    Targeted delivery of drugs and sensors into cells is an attractive technology with both medical and scientific applications. Existing delivery vehicles are generally limited by the complexity of their design, dependence on active transport, and inability to function within cellular compartments. Here, we developed self-assembled nanofibrous hydrogel fibers using a biologically inert, low-molecular-weight amphiphile. Self-assembled nanofibrous hydrogels offer unique physical/mechanical properties and can easily be loaded with a diverse range of payloads. Unlike commercially available E. coli membrane particles covalently bound to the pH reporting dye pHrodo, pHrodo encapsulated in self-assembled hydrogel-fibers internalizes into macrophages at both physiologic (37°C) and sub-physiologic (4°C) temperatures through an energy-independent, passive process. Unlike dye alone or pHrodo complexed to E. coli, pHrodo-SAFs report pH in both the cytoplasm and phagosomes, as well the nucleus. This new class of materials should be useful for next-generation sensing of the intracellular milieu. PMID:24667734

  12. Mercury-pollution induction of intracellular lipid accumulation and lysosomal compartment amplification in the benthic foraminifer Ammonia parkinsoniana

    DOE PAGESBeta

    Frontalini, Fabrizio; Curzi, Davide; Cesarini, Erica; Canonico, Barbara; Giordano, Francesco M.; De Matteis, Rita; Bernhard, Joan M.; Pieretti, Nadia; Gu, Baohua; Eskelsen, Jeremy R.; et al

    2016-09-07

    In this study, heavy metals such as mercury (Hg) pose a significant health hazard through bioaccumulation and biomagnification. By penetrating cell membranes, heavy metal ions may lead to pathological conditions. Here we examined the responses of Ammonia parkinsoniana, a benthic foraminiferan, to different concentrations of Hg in the artificial sea water. Confocal images of untreated and treated specimens using fluorescent probes (Nile Red and Acridine Orange) provided an opportunity for visualizing the intracellular lipid accumulation and acidic compartment regulation. With increased Hg over time, we observed an increased number of lipid droplets, which may have acted as a detoxifying organellemore » where Hg is sequestered and biologically inactivated. Further, Hg seems to promote the proliferation of lysosomes both in terms of number and dimension that, at the highest level of Hg, resulted in cell death. We report, for the first time, the presence of Hg within the foraminiferal cell: at the basal part of pores, in the organic linings of the foramen/septa, and as cytoplasmic accumulations.« less

  13. Mercury-Pollution Induction of Intracellular Lipid Accumulation and Lysosomal Compartment Amplification in the Benthic Foraminifer Ammonia parkinsoniana.

    PubMed

    Frontalini, Fabrizio; Curzi, Davide; Cesarini, Erica; Canonico, Barbara; Giordano, Francesco M; De Matteis, Rita; Bernhard, Joan M; Pieretti, Nadia; Gu, Baohua; Eskelsen, Jeremy R; Jubb, Aaron M; Zhao, Linduo; Pierce, Eric M; Gobbi, Pietro; Papa, Stefano; Coccioni, Rodolfo

    2016-01-01

    Heavy metals such as mercury (Hg) pose a significant health hazard through bioaccumulation and biomagnification. By penetrating cell membranes, heavy metal ions may lead to pathological conditions. Here we examined the responses of Ammonia parkinsoniana, a benthic foraminiferan, to different concentrations of Hg in the artificial sea water. Confocal images of untreated and treated specimens using fluorescent probes (Nile Red and Acridine Orange) provided an opportunity for visualizing the intracellular lipid accumulation and acidic compartment regulation. With increased Hg over time, we observed an increased number of lipid droplets, which may have acted as a detoxifying organelle where Hg is sequestered and biologically inactivated. Further, Hg seems to promote the proliferation of lysosomes both in terms of number and dimension that, at the highest level of Hg, resulted in cell death. We report, for the first time, the presence of Hg within the foraminiferal cell: at the basal part of pores, in the organic linings of the foramen/septa, and as cytoplasmic accumulations. PMID:27603511

  14. Physiological Intracellular Crowdedness is Defined by the Perimeter-to-Area Ratio of Sub-Cellular Compartments

    PubMed Central

    Hiroi, Noriko; Okuhara, Takahiro; Kubojima, Takeshi; Iba, Keisuke; Tabira, Akito; Yamashita, Shuji; Okada, Yasunori; Kobayashi, Tetsuya J.; Funahashi, Akira

    2012-01-01

    The intracellular environment is known to be a crowded and inhomogeneous space. Such an in vivo environment differs from a well-diluted, homogeneous environment for biochemical reactions. However, the effects of both crowdedness and the inhomogeneity of environment on the behavior of a mobile particle have not yet been investigated sufficiently. As described in this paper, we constructed artificial reaction spaces with fractal models, which are assumed to be non-reactive solid obstacles in a reaction space with crevices that function as operating ranges for mobile particles threading the space. Because of the homogeneity of the structures of artificial reaction spaces, the models succeeded in reproducing the physiological fractal dimension of solid structures with a smaller number of non-reactive obstacles than in the physiological condition. This incomplete compatibility was mitigated when we chose a suitable condition of a perimeter-to-area ratio of the operating range to our model. Our results also show that a simulation space is partitioned into convenient reaction compartments as an in vivo environment with the exact amount of solid structures estimated from TEM images. The characteristics of these compartments engender larger mean square displacement of a mobile particle than that of particles in smaller compartments. Subsequently, the particles start to show confined particle-like behavior. These results are compatible with our previously presented results, which predicted that a physiological environment would produce quick response and slow exhaustion reactions. PMID:22936917

  15. Essentially All Excess Fibroblast Cholesterol Moves from Plasma Membranes to Intracellular Compartments

    PubMed Central

    Lange, Yvonne; Ye, Jin; Steck, Theodore L.

    2014-01-01

    It has been shown that modestly increasing plasma membrane cholesterol beyond its physiological set point greatly increases the endoplasmic reticulum and mitochondrial pools, thereby eliciting manifold feedback responses that return cell cholesterol to its resting state. The question arises whether this homeostatic mechanism reflects the targeting of cell surface cholesterol to specific intracellular sites or its general equilibration among the organelles. We now show that human fibroblast cholesterol can be increased as much as two-fold from 2-hydroxypropyl-β-cyclodextrin without changing the size of the cell surface pool. Rather, essentially all of the added cholesterol disperses rapidly among cytoplasmic membranes, increasing their overall cholesterol content by as much as five-fold. We conclude that the level of plasma membrane cholesterol is normally at capacity and that even small increments above this physiological set point redistribute essentially entirely to intracellular membranes, perhaps down their chemical activity gradients. PMID:25014655

  16. Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment.

    PubMed

    Nikolova, Linda S; Metzstein, Mark M

    2015-11-15

    Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae. PMID:26428009

  17. LINGO-1 Protein Interacts with the p75 Neurotrophin Receptor in Intracellular Membrane Compartments*

    PubMed Central

    Meabon, James S.; De Laat, Rian; Ieguchi, Katsuaki; Wiley, Jesse C.; Hudson, Mark P.; Bothwell, Mark

    2015-01-01

    Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75NTR·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75NTR associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75NTR. Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75NTR in a manner that is difficult to reconcile with the existence of a LINGO-1·p75NTR·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75NTR·NgR complexes in the plasma membrane. PMID:25666623

  18. Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

    PubMed Central

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of recombinant protein in the intracellular compartment is critical, at this would lessen any competition in this region between the endogenous seed proteins and the introduced foreign protein. The production yields of several recombinant proteins have been greatly increased by this strategy. PMID:23563599

  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. 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

  1. 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

  2. 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

  3. Tetherin/BST-2 is essential for the formation of the intracellular virus-containing compartment in HIV-infected macrophages

    PubMed Central

    Chu, Hin; Wang, Jaang-Jiun; Qi, Mingli; Yoon, Jeong-Joong; Chen, Xuemin; Wen, Xiaoyun; Hammonds, Jason; Ding, Lingmei; Spearman, Paul

    2012-01-01

    SUMMARY HIV-1 assembly and release occurs at the plasma membrane in T lymphocytes, while intracellular sites of virus assembly or accumulation are apparent in macrophages. The host protein tetherin (BST-2) inhibits HIV release from the plasma membrane by retaining viral particles at the cell surface, but the role of tetherin at intracellular HIV assembly sites is unclear. We determined that tetherin is significantly upregulated upon macrophage infection and localizes to an intracellular virus-containing compartment (VCC). Tetherin localized at the virus-VCC membrane interface, suggesting that tetherin physically tethers virions in VCCs. Tetherin knockdown diminished and redistributed VCCs within macrophages and promoted HIV release and cell-cell transmission. The HIV Vpu protein, which downregulates tetherin from the plasma membrane, did not fully overcome tetherin-mediated restriction of particle release in macrophages. Thus, tetherin is essential for VCC formation, and may account for morphologic differences in the apparent HIV assembly sites in macrophages versus T cells. PMID:22980332

  4. Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

    PubMed Central

    Mukherjee, Debarati; Sen, Arpita; Boettner, Douglas R.; Fairn, Gregory D.; Schlam, Daniel; Bonilla Valentin, Fernando J.; Michael McCaffery, J.; Hazbun, Tony; Staiger, Chris J.; Grinstein, Sergio; Lemmon, Sandra K.; Claudio Aguilar, R.

    2013-01-01

    Summary Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking. PMID:23943876

  5. Relative acidic compartment volume as a lysosomal storage disorder–associated biomarker

    PubMed Central

    te Vruchte, Danielle; Speak, Anneliese O.; Wallom, Kerri L.; Al Eisa, Nada; Smith, David A.; Hendriksz, Christian J.; Simmons, Louise; Lachmann, Robin H.; Cousins, Alison; Hartung, Ralf; Mengel, Eugen; Runz, Heiko; Beck, Michael; Amraoui, Yasmina; Imrie, Jackie; Jacklin, Elizabeth; Riddick, Kate; Yanjanin, Nicole M.; Wassif, Christopher A.; Rolfs, Arndt; Rimmele, Florian; Wright, Naomi; Taylor, Clare; Ramaswami, Uma; Cox, Timothy M.; Hastings, Caroline; Jiang, Xuntian; Sidhu, Rohini; Ory, Daniel S.; Arias, Begona; Jeyakumar, Mylvaganam; Sillence, Daniel J.; Wraith, James E.; Porter, Forbes D.; Cortina-Borja, Mario; Platt, Frances M.

    2014-01-01

    Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency–approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders. PMID:24487591

  6. Identification and characterization of two distinct intracellular GLUT4 pools in rat skeletal muscle: evidence for an endosomal and an insulin-sensitive GLUT4 compartment.

    PubMed Central

    Aledo, J C; Lavoie, L; Volchuk, A; Keller, S R; Klip, A; Hundal, H S

    1997-01-01

    In skeletal muscle, acute insulin treatment results in the recruitment of the GLUT4 glucose transporter from intracellular vesicular structures to the plasma membrane. The precise nature of these intracellular GLUT4 stores has, however, remained poorly defined. Using an established skeletal-muscle fractionation procedure we present evidence for the existence of two distinct intracellular GLUT4 compartments. We have shown that after fractionation of crude muscle membranes on a discontinuous sucrose gradient the majority of the GLUT4 immunoreactivity was largely present in two sucrose fractions (30 and 35%, w/w, sucrose; denoted F30 and F35 respectively) containing intracellular membranes of different buoyant densities. Here we show that these fractions contained 44+/-6 and 49+/-7% of the crude membrane GLUT4 reactivity respectively, and could be further discriminated on the basis of their immunoreactivity against specific subcellular antigen markers. Membranes from the F30 fraction were highly enriched in transferrin receptor (TfR) and annexin II, two markers of the early endosome compartment, whereas they were significantly depleted of both GLUT1 and the alpha1-subunit of (Na++K+)-ATPase, two cell-surface markers. Insulin treatment resulted in a significant reduction in GLUT4 content in membranes from the F35 fraction, whereas the amount of GLUT4 in the less dense (F30) fraction remained unaffected by insulin. Immunoprecipitation of GLUT4-containing vesicles from both intracellular fractions revealed that TfR was present in GLUT4 vesicles isolated from membranes from the F30 fraction. In contrast, GLUT4 vesicles from the F35 fraction were devoid of TfR. The aminopeptidase, vp165, was present in GLUT4 vesicles from both F30 and F35; however, vesicles isolated from F30 contained over twice as much vp165 per unit of GLUT4 than those isolated from F35. The biochemical co-localization of vp165/GLUT4 was further substantiated by double-immunogold labelling of ultrathin

  7. 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

  8. 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

  9. Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris.

    PubMed

    Keizer-Gunnink, I; Vuorela, A; Myllyharju, J; Pihlajaniemi, T; Kivirikko, K I; Veenhuis, M

    2000-02-01

    It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. PMID:10686423

  10. Isotope labeling of rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses considerable challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynth...

  11. Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

    PubMed Central

    Citores, Lucía; Wesche, Jørgen; Kolpakova, Elona; Olsnes, Sjur

    1999-01-01

    Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment. PMID:10564275

  12. Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment*

    PubMed Central

    Belman, Jonathan P.; Bian, Rachel R.; Habtemichael, Estifanos N.; Li, Don T.; Jurczak, Michael J.; Alcázar-Román, Abel; McNally, Leah J.; Shulman, Gerald I.; Bogan, Jonathan S.

    2015-01-01

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake. PMID:25561724

  13. Acetylation of TUG protein promotes the accumulation of GLUT4 glucose transporters in an insulin-responsive intracellular compartment.

    PubMed

    Belman, Jonathan P; Bian, Rachel R; Habtemichael, Estifanos N; Li, Don T; Jurczak, Michael J; Alcázar-Román, Abel; McNally, Leah J; Shulman, Gerald I; Bogan, Jonathan S

    2015-02-13

    Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake. PMID:25561724

  14. On the translocation of botulinum and tetanus neurotoxins across the membrane of acidic intracellular compartments.

    PubMed

    Pirazzini, Marco; Azarnia Tehran, Domenico; Leka, Oneda; Zanetti, Giulia; Rossetto, Ornella; Montecucco, Cesare

    2016-03-01

    Tetanus and botulinum neurotoxins are produced by anaerobic bacteria of the genus Clostridium and are the most poisonous toxins known, with 50% mouse lethal dose comprised within the range of 0.1-few nanograms per Kg, depending on the individual toxin. Botulinum neurotoxins are similarly toxic to humans and can therefore be considered for potential use in bioterrorism. At the same time, their neurospecificity and reversibility of action make them excellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by a hyperactivity of peripheral nerve terminals. The complete crystallographic structure is available for some botulinum toxins, and reveals that they consist of four domains functionally related to the four steps of their mechanism of neuron intoxication: 1) binding to specific receptors of the presynaptic membrane; 2) internalization via endocytic vesicles; 3) translocation across the membrane of endocytic vesicles into the neuronal cytosol; 4) catalytic activity of the enzymatic moiety directed towards the SNARE proteins. Despite the many advances in understanding the structure-mechanism relationship of tetanus and botulinum neurotoxins, the molecular events involved in the translocation step have been only partially elucidated. Here we will review recent advances that have provided relevant insights on the process and discuss possible models that can be experimentally tested. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. PMID:26307528

  15. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  16. 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

  17. Interaptin, an Actin-binding Protein of the α-Actinin Superfamily in Dictyostelium discoideum, Is Developmentally and cAMP-regulated and Associates with Intracellular Membrane Compartments

    PubMed Central

    Rivero, Francisco; Kuspa, Adam; Brokamp, Regine; Matzner, Monika; Noegel, Angelika A.

    1998-01-01

    In a search for novel members of the α-actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 D that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD− cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location, interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton. PMID:9700162

  18. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  19. The multidrug transporter MATE1 sequesters OCs within an intracellular compartment that has no influence on OC secretion in renal proximal tubules.

    PubMed

    Martínez-Guerrero, L J; Evans, K K; Dantzler, W H; Wright, S H

    2016-01-01

    Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OC transporter (OCT)2-mediated uptake from the blood followed by apical multidrug and toxin extruder (MATE)1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H(+) exchanger. As assessed by epifluorescence microscopy, cultured Chinese hamster ovary (CHO) cells that stably expressed human MATE1 accumulated the fluorescent OC N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in human OCT2-expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC [(3)H]1-methyl-4-phenylpyridinium (MPP) from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of V-type H(+)-ATPase, and accumulation of [(3)H]MPP and [(3)H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA, suggesting that the influence of BAF was a secondary effect involving inhibition of V-type H(+)-ATPase. The accumulation of [(3)H]MPP by isolated single nonperfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [(3)H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5 μM BAF, suggesting that vesicles loaded with OCs(+) are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion. PMID:26538438

  20. A NOVEL PNYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODEL FOR DIMETHYLARSINIC ACID (DMA): THE LUNG AS A STORAGE COMPARTMENT

    EPA Science Inventory

    A NOVEL PHYSIOLOGICALLY-BASED PHARMACOKINETIC (PBPK) MODEL FOR DIMETHYLARSINIC ACID (DMA): THE LUNG AS A STORAGE COMPARTMENT. Evans, M.V., Hughes, M.F., and Kenyon, E.M. USEPA, ORD, NHEERL, RTP, NC 27711

    DMA is the major methylated metabolite of inorganic arsenic, a kno...

  1. The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization*

    PubMed Central

    Koliwer, Judith; Park, Minjong; Bauch, Carola; von Zastrow, Mark; Kreienkamp, Hans-Jürgen

    2015-01-01

    Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking. PMID:25614626

  2. MATra - Magnet Assisted Transfection: combining nanotechnology and magnetic forces to improve intracellular delivery of nucleic acids.

    PubMed

    Bertram, J

    2006-08-01

    Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules. PMID:16918404

  3. Compartments within a compartment

    PubMed Central

    Blacque, Oliver E; Sanders, Anna AWM

    2014-01-01

    The primary cilium has emerged as a hotbed of sensory and developmental signaling, serving as a privileged domain to concentrate the functions of a wide number of channels, receptors and downstream signal transducers. This realization has provided important insight into the pathophysiological mechanisms underlying the ciliopathies, an ever expanding spectrum of multi-symptomatic disorders affecting the development and maintenance of multiple tissues and organs. One emerging research focus is the subcompartmentalised nature of the organelle, consisting of discrete structural and functional subdomains such as the periciliary membrane/basal body compartment, the transition zone, the Inv compartment and the distal segment/ciliary tip region. Numerous ciliopathy, transport-related and signaling molecules localize at these compartments, indicating specific roles at these subciliary sites. Here, by focusing predominantly on research from the genetically tractable nematode C. elegans, we review ciliary subcompartments in terms of their structure, function, composition, biogenesis and relationship to human disease. PMID:24732235

  4. Intracellular Uptake and Trafficking of Difluoroboron Dibenzoylmethane-Poly(lactic acid) Nanoparticles in HeLa Cells

    PubMed Central

    Contreras, Janette; Xie, Jiansong; Chen, Yin Jie; Pei, Hua; Zhang, Guoqing; Fraser, Cassandra L.; Hamm-Alvarez, Sarah F.

    2010-01-01

    In this study, nanoparticles based on difluoroboron dibenzoylmethane-poly(lactic acid) (BF2dbmPLA) are prepared. Polylactic acid or polylactide is a commonly used degradable polymer, while the boron dye possesses a large extinction coefficient, high emission quantum yield, 2-photon absorption, and sensitivity to the surrounding environment. BF2dbmPLA exhibits molecular weight-dependent emission properties, and can be formulated as stable nanoparticles, suggesting that its unique optical properties may be useful in multiple contexts for probing intracellular environments. Here we show that BF2dbmPLA nanoparticles are internalized into cultured HeLa cells by endocytosis, and that within the cellular milieu they retain their fluorescence properties. BF2dbmPLA nanoparticles are photostable, resisting laser-induced photobleaching under conditions that destroy the fluorescence of a common photostable probe, LysoTracker™ blue. Their endocytosis is also lipid raft-dependent, as evidenced by their significant co-localization with cholera toxin B subunit in membrane compartments after uptake, and their sensitivity of uptake to methyl-β-cyclodextrin. Additionally, BF2dbmPLA nanoparticle endocytosis utilizes microtubules and actin filaments. Internalized BF2dbmPLA nanoparticles do not accumulate in acidic late endosomes and lysosomes, but within a perinuclear non-lysosomal compartment. These findings demonstrate the feasibility of using novel BF2dbmPLA nanoparticles exhibiting diverse emission properties for in situ, live cell imaging, and suggest that their endogenous uptake occurs through a lipid-raft dependent endocytosis mechanism. PMID:20420413

  5. Cation trapping by cellular acidic compartments: Beyond the concept of lysosomotropic drugs

    SciTech Connect

    Marceau, François; Bawolak, Marie-Thérèse; Lodge, Robert; Bouthillier, Johanne; Gagné-Henley, Angélique; Gaudreault, René C.; Morissette, Guillaume

    2012-02-15

    “Lysosomotropic” cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration.

  6. 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

  7. Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments

    PubMed Central

    Allen, Doug K; Laclair, Russell W; Ohlrogge, John B; Shachar-Hill, Yair

    2012-01-01

    The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state 13C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U-13C]-sucrose, [U-13C]-glucose, [U-13C]-glutamine or [U-13C]-alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography-mass spectrometry. Amino acids including alanine, glycine and serine exhibited different 13C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed. PMID:22292468

  8. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  9. Intracellular release of rapamycin from poly (lactic acid) nanospheres modifies autophagy.

    PubMed

    Nagata, Junpei; Matsui, Makoto; Tabata, Yasuhiko

    2016-09-01

    The objective of this study is to investigate the autophagy activity of cells by the intracellular release of rapamycin (Rapa) of an autophagy inducer. Rapa was incorporated into nanospheres of poly (lactic-co-glycolic acid) (PLGA) for the controlled release of Rapa. Rapa was released from the PLGA nanospheres incorporating rapamycin (Rapa-PLGA-NS) with time while the Rapa-PLGA-NS were hydrolytically degraded. When human hepatocellular carcinoma (HepG2) cells were incubated with the Rapa-PLGA-NS, the Rapa-PLGA-NS were internalized, and the intracellular concentration was maintained over four days, indicating the intracellular Rapa release. The microtubule-associated protein 1 light chain (LC3) of an autophagy marker was significantly high for the Rapa-PLGA-NS group compared with the free Rapa group even after four days incubation. In addition, intracellular harmful ubiquitinated proteins were degraded by the intracellular release of Rapa even after four days incubation in contrast to free Rapa. It is concluded that the intracellular Rapa release is effective in modulating the autophagy activity over a longer time period. PMID:27320771

  10. Compartment syndrome

    MedlinePlus

    ... caused by repetitive activities, such as running. The pressure in a compartment only increases during that activity. Compartment syndrome is most common in the lower leg and forearm. It can also occur in the hand, foot, thigh, and upper arm.

  11. Pathogenic Yersinia Promotes Its Survival by Creating an Acidic Fluid-Accessible Compartment on the Macrophage Surface

    PubMed Central

    Bahnan, Wael; Boettner, Douglas R.; Westermark, Linda; Fällman, Maria; Schesser, Kurt

    2015-01-01

    Microbial pathogens and host immune cells each initiate events following their interaction in an attempt to drive the outcome to their respective advantage. Here we show that the bacterial pathogen Yersinia pseudotuberculosis sustains itself on the surface of a macrophage by forming acidic fluid-accessible compartments that are partially bounded by the host cell plasma membrane. These Yersinia-containing acidic compartments (YACs) are bereft of the early endosomal marker EEA1 and the lysosomal antigen LAMP1 and readily form on primary macrophages as well as macrophage-like cell lines. YAC formation requires the presence of the Yersinia virulence plasmid which encodes a type III secretion system. Unexpectedly, we found that the initial formation of YACs did not require translocation of the type III effectors into the host cell cytosol; however, the duration of YACs was markedly greater in infections using translocation-competent Y. pseudotuberculosis strains as well as strains expressing the effector YopJ. Furthermore, it was in this translocation- and YopJ-dependent phase of infection that the acidic environment was critical for Y. pseudotuberculosis survival during its interaction with macrophages. Our findings indicate that during its extracellular phase of infection Y. pseudotuberculosis initiates and then, by a separate mechanism, stabilizes the formation of a highly intricate structure on the surface of the macrophage that is disengaged from the endocytic pathway. PMID:26275291

  12. Hyaluronic Acid-Based Nanocarriers for Intracellular Targeting: Interfacial Interactions with Proteins in Cancer

    PubMed Central

    Choi, Ki Young; Saravanakumar, Gurusamy; Park, Jae Hyung; Park, Kinam

    2011-01-01

    The therapeutic efficacy of most drugs is greatly depends on their ability to cross the cellular barrier and reach their intracellular target sites. To transport the drugs effectively through the cellular membrane and to deliver them into the intracellular environment, several interesting smart carrier systems based on both synthetic or natural polymers have been designed and developed. In recent years, hyaluronic acid (HA) has emerged as a promising candidate for intracellular delivery of various therapeutic and imaging agents because of its innate ability to recognize specific cellular receptors that overexpressed on diseased cells. The aim of this review is to highlight the significance of HA in cancer, and to explore the recent advances of HA-based drug carriers towards cancer imaging and therapeutics. PMID:22079699

  13. Effects of varying media, temperature, and growth rates on the intracellular concentrations of yeast amino acids.

    PubMed

    Martínez-Force, E; Benítez, T

    1995-01-01

    Variations of the yeast free amino acid pool under different culture conditions were studied in two Saccharomyces strains, the laboratory haploid strain S288C and the industrial fermentative yeast IFI256. The internal amino acid pool of both strains was measured when grown in laboratory (minimal and complete) versus semiindustrial (molasses with or without added biotin and/or diammonium phosphate) media, in fermentable (glucose, fructose, sucrose) versus respirable (glycerol) carbon sources, in different temperatures (22, 30, and 37 degrees C), pHs (2.0-4.75), and growth rates (0.018-0.24 h-1) in continuous culture, and at different phases of the growth curve in batch culture (lag, exponential, early and late stationary). Results indicated that environmental conditions, particularly the presence of amino acids in the media, enormously influenced the intracellular amino acid concentration. Higher values were detected in molasses than in laboratory media and in fermentable carbon sources (glucose, fructose, sucrose) than in glycerol. Variations in the amino acid pool along the growth curve were greater at 37 degrees C than at other temperatures; in all cases, the highest values were measured at the beginning of the exponential phase. In continuous culture and at different growth rates, intracellular free amino acid concentrations increased by 3-10-fold when the growth rate was lower than 0.05 h-1, representing 20-35% of the total (free plus protein) amino acid content and indicating that amino acid yield was a partly growth-linked parameter. PMID:7654310

  14. Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue.

    PubMed

    Foster, J G; Cress, W D; Wright, S F; Hess, J L

    1987-04-01

    The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var ;Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells. PMID:16665360

  15. Intracellular pH Recovery Rates in Bivalve Hemocytes Following Exposure to Acidic Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Croxton, A.; Wikfors, G. H.

    2012-12-01

    Predictions of ocean acidification effects upon carbonate shell-forming species have caused great concern for the future of shellfisheries. Nevertheless, bivalve species inhabiting an estuarine environment have evolved in these environments with fluctuating pH levels. Previous experimental studies conducted in our laboratory have demonstrated the ability of oyster hemocytes to maintain intracellular homeostasis under acidic external conditions. However, little information is known of this homeostatic mechanism in other molluscan shellfish species present in these same habitats. In the current study we propose to determine if other bivalve species of aquaculture interest also possess this intracellular regulation by applying an in vitro hemocyte pH-recovery assay, previously developed for oysters, on the northern quahog, Mercenaria mercenaria, the blue mussel, Mytilus edulis, and the softshell clam, Mya arenaria. Preliminary results from the determination of initial intracellular pH levels, the initial step in the rate recovery assay, indicated a pH range between 7.0-7.4. This range was comparable to initial values measured in oysters, and consistent with data reported in the current literature. The second step of the hemocyte pH-recovery assay involves exposing oyster hemocytes to acidic external conditions and measuring the ability of the hemocyte intracellular pH to maintain homeostasis (i.e. recovery rate). Results from the recovery rate process will be presented.

  16. Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

    PubMed Central

    Patel, Sandip; Docampo, Roberto

    2010-01-01

    Changes in cytosolic calcium concentration are crucial for a variety of cellular processes in all cells. It has long been appreciated that calcium is stored and released from intracellular calcium stores such as the endoplasmic reticulum. However, emerging evidence indicates that calcium is also dynamically regulated by a seemingly disparate collection of acidic organelles. Here, we review the defining features of these acidic calcium stores and highlight recent progress in understanding the mechanisms of uptake and release of calcium from these stores. We also examine the nature of calcium buffering within the stores and summarize the physiological and patho-physiological significance of these ubiquitous organelles in calcium signaling. PMID:20303271

  17. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy).

    PubMed

    Amato, Alberto; Lüdeking, Alexander; Kooistra, Wiebe H C F

    2010-01-01

    Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents. PMID:19615395

  18. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  19. 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

  20. Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis

    SciTech Connect

    Nitschke, Matthias; Korte, Thomas; Ter-Avetisyan, Gohar; Tuennemann, Gisela; Cardoso, M. Cristina; Veit, Michael Herrmann, Andreas

    2008-08-01

    Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kindey cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.

  1. Drug resistance to paclitaxel is not only associated with ABCB1 mRNA expression but also with drug accumulation in intracellular compartments in human lung cancer cell lines.

    PubMed

    Shimomura, Masanori; Yaoi, Takeshi; Itoh, Kyoko; Kato, Daishiro; Terauchi, Kunihiko; Shimada, Junichi; Fushiki, Shinji

    2012-04-01

    In order to clarify the mechanisms of resistance to paclitaxel in lung cancer, three human lung cancer cell lines which exhibit different sensitivity to paclitaxel were investigated from the following viewpoints: overexpression of ATP-binding cassette, sub-family B, member 1 (ABCB1), mutations on paclitaxel binding site of β-tubulin genes, quantity of polymerized tubulin and the intracellular localization of paclitaxel. ABCB1 expression was evaluated by real-time RT-PCR. No correlations were noted between the ABCB1 expression in the sensitive and resistant cell lines at the mRNA level. No mutations on the paclitaxel binding site of the β-tubulin genes were detected in either the resistant or sensitive cells. Live cell images obtained by confocal laser microscopy revealed that the resistant cell line, RERF-LC-KJ, had more accumulation of Oregon Green® 488 conjugated paclitaxel in the lysosomal and extra-lysosomal compartments of cytoplasm than other cell lines. The results obtained in this study indicated that the changes in the subcellular localization could contribute to the production of paclitaxel resistance in lung cancer cell lines. Further studies should be conducted to elucidate the molecular mechanisms that differentiate the intracellular localization of paclitaxel. PMID:22179563

  2. Drug resistance to paclitaxel is not only associated with ABCB1 mRNA expression but also with drug accumulation in intracellular compartments in human lung cancer cell lines

    PubMed Central

    SHIMOMURA, MASANORI; YAOI, TAKESHI; ITOH, KYOKO; KATO, DAISHIRO; TERAUCHI, KUNIHIKO; SHIMADA, JUNICHI; FUSHIKI, SHINJI

    2012-01-01

    In order to clarify the mechanisms of resistance to paclitaxel in lung cancer, three human lung cancer cell lines which exhibit different sensitivity to paclitaxel were investigated from the following viewpoints: overexpression of ATP-binding cassette, sub-family B, member 1 (ABCB1), mutations on paclitaxel binding site of β-tubulin genes, quantity of polymerized tubulin and the intracellular localization of paclitaxel. ABCB1 expression was evaluated by real-time RT-PCR. No correlations were noted between the ABCB1 expression in the sensitive and resistant cell lines at the mRNA level. No mutations on the paclitaxel binding site of the β-tubulin genes were detected in either the resistant or sensitive cells. Live cell images obtained by confocal laser microscopy revealed that the resistant cell line, RERF-LC-KJ, had more accumulation of Oregon Green® 488 conjugated paclitaxel in the lysosomal and extra-lysosomal compartments of cytoplasm than other cell lines. The results obtained in this study indicated that the changes in the subcellular localization could contribute to the production of paclitaxel resistance in lung cancer cell lines. Further studies should be conducted to elucidate the molecular mechanisms that differentiate the intracellular localization of paclitaxel. PMID:22179563

  3. Compartment syndrome

    MedlinePlus

    ... Jobe MT. Compartment syndromes and Volkmann contracture. In: Canale ST, Beaty JH, eds. Campbell's Operative Orthopaedics . 12th ed. ... and Shoulder Service, UCSF Department of Orthopaedic Surgery, San Francisco, CA. Also reviewed by David Zieve, MD, MHA, ...

  4. Compartment syndromes

    NASA Technical Reports Server (NTRS)

    Mubarak, S. J.; Pedowitz, R. A.; Hargens, A. R.

    1989-01-01

    The compartment syndrome is defined as a condition in which high pressure within a closed fascial space (muscle compartment) reduces capillary blood perfusion below the level necessary for tissue viability'. This condition occurs in acute and chronic (exertional) forms, and may be secondary to a variety of causes. The end-result of an extended period of elevated intramuscular pressure may be the development of irreversible tissue injury and Volkmann's contracture. The goal of treatment of the compartment syndrome is the reduction of intracompartmental pressure thus facilitating reperfusion of ischaemic tissue and this goal may be achieved by decompressive fasciotomy. Controversy exists regarding the critical pressure-time thresholds for surgical decompression and the optimal diagnostic methods of measuring intracompartmental pressures. This paper will update and review some current knowledge regarding the pathophysiology, aetiology, diagnosis, and treatment of the acute compartment syndrome.

  5. 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

  6. 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

  7. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

    PubMed Central

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M.; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  8. Cathepsin B-sensitive polymers for compartment-specific degradation and nucleic acid release

    PubMed Central

    Chu, David S.H.; Johnson, Russell N.; Pun, Suzie H.

    2011-01-01

    Degradable cationic polymers are desirable for in vivo nucleic acid delivery because they offer significantly decreased toxicity over non-degradable counterparts. Peptide linkers provide chemical stability and high specificity for particular endopeptidases but have not been extensively studied for nucleic acid delivery applications. In this work, enzymatically degradable peptide-HPMA copolymers were synthesized by RAFT polymerization of HPMA with methacrylated peptide macromonomers, resulting in polymers with low polydispersity and near quantitative incorporation of peptides. Three peptide-HPMA copolymers were evaluated: (i) pHCathK10, containing peptides composed of the linker phe-lys-phe-leu (FKFL), a substrate of the endosomal/lysosomal endopeptidase cathepsin B, connected to oligo-(l)-lysine for nucleic acid binding, (ii) pHCath(d)K10, containing the FKFL linker with oligo-(d)-lysine, and (iii) pH(d)Cath(d)K10, containing all (d) amino acids. Cathepsin B degraded copolymers pHCathK10 and pHCath(d)K10 within one hour while no degradation of pH(d)Cath(d)K10 was observed. Polyplexes formed with pHCathK10 copolymers show DNA release by 4 hrs of treatment with cathepsin B; comparatively, polyplexes formed with pHCath(d)K10 and pH(d)Cath(d)K10 show no DNA release within 8 hrs. Transfection efficiency in HeLa and NIH/3T3 cells were comparable between the copolymers but pHCathK10 was less toxic. This work demonstrates the successful application of peptide linkers for degradable cationic polymers and DNA release. PMID:22036879

  9. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  10. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

    PubMed

    Shane, Michael W; Stigter, Kyla; Fedosejevs, Eric T; Plaxton, William C

    2014-11-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  11. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

    NASA Astrophysics Data System (ADS)

    Chiappini, C.; De Rosa, E.; Martinez, J. O.; Liu, X.; Steele, J.; Stevens, M. M.; Tasciotti, E.

    2015-05-01

    The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

  12. Gold nanoparticles with different amino acid surfaces: serum albumin adsorption, intracellular uptake and cytotoxicity.

    PubMed

    Cai, Huanxin; Yao, Ping

    2014-11-01

    Gold nanoparticles with aspartate, glycine, leucine, lysine, and serine surfaces were produced from the mixed solutions of HAuCl4 and respective amino acids via UV irradiation. The amino acids bind to the nanoparticle surfaces via amine groups and their carboxylic groups extend out to stabilize the nanoparticles. The nanoparticles have diameters of 15-47 nm in pH 7.4 aqueous solution and have diameters of 62-73 nm after 48 h incubation in cell culture containing serum. The nanoparticles adsorb human and bovine serum albumins on their surfaces by specific interactions, characterized by the intrinsic fluorescence quenching of the albumins. The albumin adsorption effectively decreases the aggregation of the nanoparticles in cell culture and also decreases the intracellular uptake of the nanoparticles. The gold nanoparticles produced from leucine and lysine, which have amphiphilic groups on their surfaces, present better biocompatibility than the other gold nanoparticles. PMID:25466455

  13. Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro.

    PubMed

    Yajima, T

    1986-01-01

    Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues. PMID:3742560

  14. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  15. The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of Aspergillus niger through Intracellular Acidification

    PubMed Central

    Plumridge, Andrew; Hesse, Stephan J. A.; Watson, Adrian J.; Lowe, Kenneth C.; Stratford, Malcolm; Archer, David B.

    2004-01-01

    The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 105/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pHcyt) by more than 1 pH unit and a depression of vacuolar pH (pHvac) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pHcyt. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth. PMID:15184150

  16. Cell nucleus targeting for living cell extraction of nucleic acid associated proteins with intracellular nanoprobes of magnetic carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Liu, Fangjie; Cheng, Kai; Wu, Ren'an; Zou, Hanfa

    2013-08-01

    Since nanoparticles could be ingested by cells naturally and target at a specific cellular location as designed, the extraction of intracellular proteins from living cells for large-scale analysis by nanoprobes seems to be ideally possible. Nucleic acid associated proteins (NAaP) take the crucial position during biological processes in maintaining and regulating gene structure and gene related behaviors, yet there are still challenges during the global investigation of intracellular NAaP, especially from living cells. In this work, a strategy to extract intracellular proteins from living cells with the magnetic carbon nanotube (oMWCNT@Fe3O4) as an intracellular probe is developed, to achieve the high throughput analysis of NAaP from living human hepatoma BEL-7402 cells with a mass spectrometry-based proteomic approach. Due to the specific intracellular localization of the magnetic carbon nanotubes around nuclei and its strong interaction with nucleic acids, the highly efficient extraction was realized for cellular NAaP from living cells, with the capability of identifying 2383 intracellular NAaP from only ca. 10,000 living cells. This method exhibited potential applications in dynamic and in situ analysis of intracellular proteins. PMID:23815738

  17. Question 7: biosynthesis of phosphatidic acid in liposome compartments - toward the self-reproduction of minimal cells.

    PubMed

    Kuruma, Yutetsu

    2007-10-01

    Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive 'synthetic cells (minimal cells)' approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes - like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. PMID:17653611

  18. 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

  19. Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification.

    PubMed Central

    Holmes, Melanie E; Mwanjewe, James; Samson, Sue E; Haist, James V; Wilson, John X; Dixon, S Jeffrey; Karmazyn, Morris; Grover, Ashok K

    2002-01-01

    Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion. PMID:11853561

  20. Effects of Fatty Acids on Intracellular [Ca2+], Mitochondrial Uncoupling and Apoptosis in Rat Pachytene Spermatocytes and Round Spermatids

    PubMed Central

    Paillamanque, Joaquín; Madrid, Cristian; Carmona, Emerson M.; Osses, Nelson; Moreno, Ricardo D.; Oresti, Gerardo M.; Pino, José A.

    2016-01-01

    The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation. PMID:27428262

  1. Bile Acids Regulate Nuclear Receptor (Nur77) Expression and Intracellular Location to Control Proliferation and Apoptosis

    PubMed Central

    Hu, Ying; Chau, Thinh; Liu, Hui-xin; Liao, Degui; Keane, Ryan; Nie, Yuqiang; Yang, Hui; Wan, Yu-Jui Yvonne

    2014-01-01

    Bile acids (BAs) are endogenous agents capable of causing cancer throughout the gastrointestinal (GI) tract. To uncover the mechanism by which BAs exert carcinogenic effects, both human liver and colon cancer cells as well as mouse primary hepatocytes were treated with BAs and assayed for viability, genotoxic stress, and transcriptional response. BAs induced both Nur77 (NR4A1) and pro-inflammatory gene expression. The intracellular location of BA-induced Nur77 was time-dependent; short-term (1–3 h) exposure induced nuclear Nur77 whereas longer (1–2 days) exposure also increased cytosolic Nur77 expression and apoptosis. Inhibiting Nur77 nuclear export with leptomycin B decreased LCA-induced apoptosis. Extended (7 days) treatment with BA generated resistance to BA with increased nuclear Nur77, viability, and mobility. While, knockdown of Nur77 in BA-resistant cells increased cellular susceptibility to LCA-induced apoptosis. Moreover, in vivo mouse xenograft experiments demonstrated that BA-resistant cells form larger tumors with elevated Nur77 expression compared to parental controls. DNA-binding and gene expression assays identified multiple survival genes (CDK4, CCND2, MAP4K5, STAT5A, and RBBP8) and a pro-apoptosis gene (BID) as Nur77 targets. Consistently, BA-induced up-regulation of the aforementioned genes was abrogated by a lack of Nur77. Importantly, Nur77 was overexpressed in high percentage of human colon and liver cancer specimens and the intracellular location of Nur77 correlated with elevated serum total BA levels in colon cancer patients. These data show for the first time that BAs via Nur77 have a dual role in modulating cell survival and death. Implications: These findings establish a direct link between Nur77 and the carcinogenic effect of bile acids. PMID:25232032

  2. Spherical Nucleic Acids as Intracellular Agents for Nucleic Acid Based Therapeutics

    NASA Astrophysics Data System (ADS)

    Hao, Liangliang

    Recent functional discoveries on the noncoding sequences of human genome and transcriptome could lead to revolutionary treatment modalities because the noncoding RNAs (ncRNAs) can be applied as therapeutic agents to manipulate disease-causing genes. To date few nucleic acid-based therapeutics have been translated into the clinic due to challenges in the delivery of the oligonucleotide agents in an effective, cell specific, and non-toxic fashion. Unmodified oligonucleotide agents are destroyed rapidly in biological fluids by enzymatic degradation and have difficulty crossing the plasma membrane without the aid of transfection reagents, which often cause inflammatory, cytotoxic, or immunogenic side effects. Spherical nucleic acids (SNAs), nanoparticles consisting of densely organized and highly oriented oligonucleotides, pose one possible solution to circumventing these problems in both the antisense and RNA interference (RNAi) pathways. The unique three dimensional architecture of SNAs protects the bioactive oligonucleotides from unspecific degradation during delivery and supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. Owing to their unique structure, SNAs are able to cross cell membranes and regulate target genes expression as a single entity, without triggering the cellular innate immune response. Herein, my thesis has focused on understanding the interactions between SNAs and cellular components and developing SNA-based nanostructures to improve therapeutic capabilities. Specifically, I developed a novel SNA-based, nanoscale agent for delivery of therapeutic oligonucleotides to manipulate microRNAs (miRNAs), the endogenous post-transcriptional gene regulators. I investigated the role of SNAs involving miRNAs in anti-cancer or anti-inflammation responses in cells and in in vivo murine disease models via systemic injection. Furthermore, I explored using different strategies to construct

  3. Intracellular pH Recovery Rates of Hemocytes from Estuarine and Open Ocean Bivalve Species Following In vitro Acid Challenge

    NASA Astrophysics Data System (ADS)

    Croxton, A.; Wikfors, G.

    2013-12-01

    Decreasing pH in estuarine systems is a growing concern for researchers studying mollusk species. Debates continue on whether estuarine bivalve species are more or less vulnerable to ocean acidification than marine species because estuaries can present multiple environmental stressors. The aim of this study is to understand the homeostatic mechanisms of bivalve hemocytes following exposure to extracellular acid treatment. Previous measurements using fluorescent SNARF probes and flow-cytometry have determined the intracellular pH of hemocytes from several bivalve species (eastern oyster, bay scallop, northern quahog, soft-shell clam, and blue mussel) to range between 7.0-7.4. In the present study of four bivalve species, recovery rate profiles were determined for intracellular hemocyte pH following addition of acid to hemolymph in vitro. These profiles indicate that soft-shell clams and bay scallops maintained homeostasis with very little change in intracellular pH. In contrast, an initial drop in intracellular pH in northern quahogs was followed by a steady recovery of intracellular pH. Contrasting results between species appear to be unrelated to mineral shell composition (aragonite vs. calcite) or habitat location (infaunal vs. epifaunal). The next phase of this study will be to determine if offshore species (surfclams and sea scallops) will have similar responses. Results from these studies will provide a better understanding of the physiological responses of estuarine and marine species exposed to acidified environments.

  4. Sequestration of AS-DACA into Acidic Compartments of the Membrane Trafficking System as a Mechanism of Drug Resistance in Rhabdomyosarcoma

    PubMed Central

    Williams, Marissa; Catchpoole, Daniel

    2013-01-01

    The accumulation of weakly basic drugs into acidic organelles has recently been described as a contributor to resistance in childhood cancer rhabdomyosarcoma (RMS) cell lines with differential sensitivity to a novel topoisomerase II inhibitor, AS-DACA. The current study aims to explore the contribution of the endocytic pathway to AS-DACA sequestration in RMS cell lines. A 24-fold differential in AS-DACA cytotoxicity was detected between the RMS lines RD and Rh30. The effect of inhibitors of the endocytic pathway on AS-DACA sensitivity in RMS cell lines, coupled with the variations of endosomal marker expression, indicated the late endosomal/lysosomal compartment was implicated by confounding lines of evidence. Higher expression levels of Lysosomal-Associated Membrane Protein-1 (LAMP1) in the resistant RMS cell line, RD, provided correlations between the increased amount and activity of these compartments to AS-DACA resistance. The late endosomal inhibitor 3-methyladenine increased AS-DACA sensitivity solely in RD leading to the reduction of AS-DACA in membrane trafficking organelles. Acidification inhibitors did not produce an increase in AS-DACA sensitivity nor reduce its sequestration, indicating that the pH partitioning of weakly basic drugs into acidic compartments does not likely contribute to the AS-DACA sequestering resistance mechanism evident in RMS cells. PMID:23799359

  5. A novel glyceryl monoolein-bearing cubosomes for gambogenic acid: Preparation, cytotoxicity and intracellular uptake.

    PubMed

    Luo, Qing; Lin, Tongyuan; Zhang, Cai Yuan; Zhu, Tingting; Wang, Lei; Ji, Zhaojie; Jia, Buyun; Ge, Tao; Peng, Daiyin; Chen, Weidong

    2015-09-30

    Lyotropic cubic liquid crystalline nanoparticles, also known as 'cubosomes', have been tested as effective carriers for a variety of drugs due to their ability to enhance delivery efficiency and reduced drug side effects. Cubosomes are colloidal carriers composed of biodegradable Glyceryl monooleate and F127. Being composed of well tolerable and physiological materials, these carriers are well tolerated, compatible and non-toxic. In this study, therefore, we developed a novel, water-soluble, glyceryl monooleate and F127 based multiblock copolymer for Gambogenic acid (GNA) by emulsion-evaporation and low temperature-solidification technique. Physicochemical properties, in vitro cytotoxicity, cellular uptake and in vivo pharmacokinetic of GNA-loaded cubosomes (GNA-Cubs) were investigated. The results revealed that GNA-Cubs were spherical or ellipsoidal monocellular by dynamic light scattering, meanwhile, 150-250nm in mean size with narrow polydispersity indexas determined by transmission electron microscopy. Small angle X-ray scattering indicated that GNA-Cubs retain the Pn3m cubic symmetry. Compared with GNA solution, GNA-Cubs exhibited markedly prolonged inhibitory activity in SMMC-7721 cells, as well as time-dependent increases in intra-cellular uptake. Furthermore, in vivo pharmacokinetic study showed that the Cmax values and the AUC of GNA-Cubs were higher than GNA solution approximately 1.2-fold and 9.1-fold, respectively. In conclusion, the results showed that the cubic liquid crystalline nanoparticles could be a potentially nanocarrier in the delivery of GNA for cancer therapy. PMID:26209071

  6. Acidic intracellular pH shift during Caenorhabditis elegans larval development

    SciTech Connect

    Wadsworth, W.G.; Riddle, D.L. )

    1988-11-01

    During recovery from the developmentally arrested, nonfeeding dauer stage of the nemotode Caenorhabditis elegans, metabolic activation is accompanied by a decrease in intracellular pH (pH{sub i}). Phosphorus-31 nuclear magnetic resonance ({sup 31}P NMR) analyses of perchloric acid extracts show that inorganic phosphate predominates in dauer larvae, whereas ATP and other high-energy metabolites are abundant within 6 hr after dauer larvae have been placed in food to initiate development. Although metabolic activation has been associated with an alkaline pH{sub i} shift in other organisms, in vivo {sup 31}P NMR analysis of recovering dauer larvae shows a pH{sub i} decrease from {approx} 7.3 to {approx} 6.3 within 3 hr after the animals encounter food. This shift occurs before feeding begins, and it coincides with, or soon follows, the developmental commitment to recover from the dauer stage, suggesting that control of pH{sub i} may be important in the regulation of larval development in nematodes.

  7. Bacteriophage SP82G Inhibition of an Intracellular Deoxyribonucleic Acid Inactivation Process in Bacillus subtilis1

    PubMed Central

    McAllister, William T.; Green, D. MacDonald

    1972-01-01

    The stability of SP82G bacteriophage deoxyribonucleic acid (DNA) after its uptake by competent Bacillus subtilis was examined by determining the ability of superinfecting phage particles to rescue genetic markers carried by the infective DNA. These experiments show that a DNA inactivation process within the cell is inhibited after infection of the cell by intact phage particles. The inhibition is maximally expressed 6 min after phage infection and is completely prevented by the addition of chloramphenicol at the time of infection. The protective effect of this function extends even to infective DNA which was present in the cell before the addition of intact phage. Continued protein synthesis does not appear to be a requirement for the maintenance of the inhibition. In an analogous situation, if infectious centers resulting from singly infecting phage particles are exposed to chloramphenicol shortly after the time of infection, an exponential decrease in the survival of infectious centers with time held in chloramphenicol is observed. If the addition of chloramphenicol is delayed until 6 min after infection, the infectious centers are resistant to chloramphenicol. The sensitivity of infectious centers treated with chloramphenicol at early times after infection is strongly dependent upon the multiplicity of infection and is consistent with a model of multiplicity reactivation. These results indicate that injected DNA is also susceptible to the intracellular inactivation process and suggest that the inhibition of this system is necessary for the successful establishment of an infectious center. PMID:4625174

  8. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels.

    PubMed

    Lee, Hyukjin; Mok, Hyejung; Lee, Soohyeon; Oh, Yu-Kyoung; Park, Tae Gwan

    2007-06-01

    Novel hyaluronic acid (HA) nanogels physically encapsulating small interfering RNA (siRNA) were fabricated by an inverse water-in-oil emulsion method. Thiol-conjugated HA dissolved in aqueous emulsion droplets was ultrasonically crosslinked via the formation of disulfide linkages to produce HA nanogels with a size distribution from 200 to 500 nm. Green fluorescence protein (GFP) siRNA was physically entrapped within the HA nanogels during the emulsion/crosslinking process. The HA/siRNA nanogels were readily taken up by HA receptor positive cells (HCT-116 cells) having HA-specific CD44 receptors on the surface. Release rates of siRNA from the HA nanogels could be modulated by changing the concentration of glutathione (GSH) in the buffer solution, indicating that the degradation/erosion of disulfide crosslinked HA nanogels, triggered by an intracellular reductive agent, controlled the release pattern of siRNA. When HA nanogels containing GFP siRNA were co-transfected with GFP plasmid/Lipofectamine to HCT-116 cells, a significant extent of GFP gene silencing was observed in both serum and non-serum conditions. The gene silencing effect was reduced in the presence of free HA in the transfection medium, revealing that HA nanogels were selectively taken up by HCT-116 cells via receptor mediated endocytosis. PMID:17408798

  9. Effect of inhibitors of arachidonic acid metabolism on efflux of intracellular enzymes from skeletal muscle following experimental damage.

    PubMed Central

    Jackson, M J; Wagenmakers, A J; Edwards, R H

    1987-01-01

    The role of arachidonic acid metabolism in the efflux of intracellular enzymes from damaged skeletal muscle has been examined in vitro using inhibitors of cyclo-oxygenase and lipoxygenase enzymes. Damage to skeletal muscle induced by either calcium ionophore A23187 (25 microM) or dinitrophenol (1 mM) caused an increase in the efflux of prostaglandins E2 and F2 alpha together with a large efflux of intracellular creatine kinase. Use of a cyclo-oxygenase inhibitor completely prevented the efflux of prostaglandins, but had no effect on creatine kinase efflux. However, several agents having the ability to inhibit lipoxygenase enzymes dramatically reduced creatine kinase efflux following damage. These data suggest that a product or products of lipoxygenase enzymes may be mediators of the changes in plasma membrane integrity which permit efflux of intracellular enzymes as a consequence of skeletal muscle damage. PMID:3109374

  10. Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

    PubMed

    Kwak, Min-Kyu; Song, Sung-Hyun; Ku, MyungHee; Kang, Sa-Ouk

    2015-07-01

    Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression. PMID:25957768

  11. Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: role of glycine conjugates.

    PubMed

    Chatterjee, Sagnik; Bijsmans, Ingrid T G W; van Mil, Saskia W C; Augustijns, Patrick; Annaert, Pieter

    2014-03-01

    Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5-4h) to 5-100μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH. PMID:24211540

  12. pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery

    NASA Astrophysics Data System (ADS)

    Li, Shengliang; Hu, Kelei; Cao, Weipeng; Sun, Yun; Sheng, Wang; Li, Feng; Wu, Yan; Liang, Xing-Jie

    2014-10-01

    To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a phenylboronic acid-modified poly(lactic acid)-poly(ethyleneimine)(PLA-PEI) copolymer loaded with doxorubicin (Dox) for intracellular imaging and pH-responsive drug delivery. The nanoparticles exhibited superior fluorescence properties, such as fluorescence stability, no blinking and excitation-dependent fluorescence behavior. The Dox-loaded fluorescent nanoparticles showed pH-responsive drug release and were more effective in suppressing the proliferation of MCF-7 cells. In addition, the biocompatible fluorescent nanoparticles could be used as a tool for intracellular imaging and drug delivery, and the process of endosomal escape was traced by real-time imaging. These pH-responsive and biocompatible fluorescent polymer nanoparticles, based on phenylboronic acid, are promising tools for intracellular imaging and drug delivery.To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a

  13. Amphipathic β2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

    PubMed Central

    Tiirola, Terttu M.; Strøm, Morten B.; Vuorela, Pia M.

    2016-01-01

    We demonstrate in the current work that small cationic antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 μM, i.e. < 3.4 μg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the β2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these β2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic β2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen. PMID:27280777

  14. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

    PubMed

    Bates, Ryan C; Fees, Colby P; Holland, William L; Winger, Courtney C; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J

    2014-02-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  15. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism

    PubMed Central

    Toledo, Daniel A. M.; Roque, Natália R.; Teixeira, Lívia; Milán-Garcés, Erix A.; Carneiro, Alan B.; Almeida, Mariana R.; Andrade, Gustavo F. S.; Martins, Jefferson S.; Pinho, Roberto R.; Freire-de-Lima, Célio G.; Bozza, Patrícia T.; D’Avila, Heloisa

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas’ disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  16. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism.

    PubMed

    Toledo, Daniel A M; Roque, Natália R; Teixeira, Lívia; Milán-Garcés, Erix A; Carneiro, Alan B; Almeida, Mariana R; Andrade, Gustavo F S; Martins, Jefferson S; Pinho, Roberto R; Freire-de-Lima, Célio G; Bozza, Patrícia T; D'Avila, Heloisa; Melo, Rossana C N

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  17. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  18. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

    SciTech Connect

    Louisse, Jochem; Verwei, Miriam; Sandt, Johannes J.M. van de; Rietjens, Ivonne M.C.M.

    2010-06-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH{sub i}) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH{sub i}in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH{sub i} of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na{sup +}/H{sup +}-antiporter, corroborating an important role of the pH{sub i} in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH{sub i} may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  19. Novel pH-sensitive polysialic acid based polymeric micelles for triggered intracellular release of hydrophobic drug.

    PubMed

    Zhang, Wuxia; Dong, Dongqi; Li, Peng; Wang, Dongdong; Mu, Haibo; Niu, Hong; Duan, Jinyou

    2016-03-30

    Polysialic acid (PSA), a non-immunogenic and biodegradable natural polymer, is prone to hydrolysis under endo-lysosomal pH conditions. Here, we synthesized an intracellular pH-sensitive polysialic acid-ursolic acid conjugate by a condensation reaction. To further test the drug loading capability, we prepared paclitaxel-loaded polysialic acid-based amphiphilic copolymer micelle (PTX-loaded-PSAU) by a nanoprecipitation method. Results showed PTX-loaded-PSAU exhibited well-defined spherical shape and homogeneous distribution. The drug-loading was 4.5% with an entrapment efficiency of 67.5%. PTX released from PTX-loaded-PSAU was 15% and 42% in 72 h under simulated physiological condition (pH 7.4) and mild acidic conditions (pH 5.0), respectively. In addition, In vitro cytotoxicity assay showed that PTX-loaded-PSAU retained anti-tumor (SGC-7901) activity with a cell viability of 53.8% following 72 h incubation, indicating PTX-loaded-PSAU could efficiently release PTX into the tumor cells. These results indicated that the pH-responsive biodegradable PTX-loaded-PSAU possess superior extracellular stability and intracellular drug release ability. PMID:26794949

  20. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  1. Localization of CdSe/ZnS quantum dots in the lysosomal acidic compartment of cultured neurons and its impact on viability: potential role of ion release.

    PubMed

    Corazzari, Ingrid; Gilardino, Alessandra; Dalmazzo, Simona; Fubini, Bice; Lovisolo, Davide

    2013-03-01

    CdSe Quantum Dots (QDs) are increasingly being employed in both industrial applications and biological imaging, thanks to their numerous advantages over conventional organic and proteic fluorescent markers. On the other hand a growing concern has emerged that toxic elements from the QDs core would render the nanoparticles harmful to cell cultures, animals and humans. The interaction between QDs and neuronal cells in particular needs to be carefully evaluated, since nanoparticles could access the nervous system by several pathways, including the olfactory epithelium, even if no data are presently available about QDs. The pH of the environment to which the nanoparticles are exposed may play a crucial role in the stability of QDs coating. For this reason we investigated the release of metal ions from CdSe/ZnS QDs in artificial media reproducing the cytosolic and lysosomal cellular compartments characterized respectively by a neutral and an acidic pH. In the latter significant amounts of both Cd(2+) and Zn(2+) were released. We provide evidence that these QDs are internalized in the GT1-7 neuronal cell line and located in the lysosomal compartment. These findings can be related to a slight but significant reduction in cell survival and proliferation. PMID:23274769

  2. 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

  3. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    PubMed

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. PMID:25554489

  4. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level

    PubMed Central

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  5. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

    PubMed

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  6. Life at high salt concentrations, intracellular KCl concentrations, and acidic proteomes

    PubMed Central

    Oren, Aharon

    2013-01-01

    Extremely halophilic microorganisms that accumulate KCl for osmotic balance (the Halobacteriaceae, Salinibacter) have a large excess of acidic amino acids in their proteins. This minireview explores the occurrence of acidic proteomes in halophiles of different physiology and phylogenetic affiliation. For fermentative bacteria of the order Halanaerobiales, known to accumulate KCl, an acidic proteome was predicted. However, this is not confirmed by genome analysis. The reported excess of acidic amino acids is due to a high content of Gln and Asn, which yield Glu and Asp upon acid hydrolysis. The closely related Halorhodospira halophila and Halorhodospira halochloris use different strategies to cope with high salt. The first has an acidic proteome and accumulates high KCl concentrations at high salt concentrations; the second does not accumulate KCl and lacks an acidic proteome. Acidic proteomes can be predicted from the genomes of some moderately halophilic aerobes that accumulate organic osmotic solutes (Halomonas elongata, Chromohalobacter salexigens) and some marine bacteria. Based on the information on cultured species it is possible to understand the pI profiles predicted from metagenomic data from hypersaline environments. PMID:24204364

  7. 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

  8. Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers.

    PubMed

    McErlean, Emma M; McCrudden, Cian M; McCarthy, Helen O

    2016-09-01

    The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration. PMID:27582234

  9. d-Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen

    PubMed Central

    2012-01-01

    Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo. PMID:23240806

  10. Elaidate, an 18-Carbon Trans-monoenoic Fatty Acid, but not Physiological Fatty Acids Increases Intracellular Zn2+ in Human Macrophages

    PubMed Central

    Zacherl, Janelle R.; Tourkova, Irina; St Croix, Claudette M.; Robinson, Lisa J.; Peck Palmer, Octavia M.; Mihalik, Stephanie J.; Blair, Harry C.

    2015-01-01

    Artificial trans fatty acids promote atherosclerosis by blocking macrophage clearance of cell debris. Classical fatty-acid response mechanisms include TLR4-NF-κB activation, and Erk1/2 phosphorylation, but these may not indicate long-term mechanisms. Indeed, nuclear NF-κB was increased by 60 minute treatment by 30 μM of the 18 carbon trans unsaturated fatty acid elaidic acid (elaidate), the physiological cis-unsaturated fatty acid oleic acid (oleate), and the 18 or 16 carbon saturated fatty acids stearic and palmitic acid (stearate or palmitate). However, except for stearate, effects on related pathways were minimal at 44 hours. To determine longer term effects of trans fatty acids, we compared whole exome mRNA expression of (trans) elaidate to (cis) oleate, 30 μM, at 44 hours in human macrophages. We found that elaidate changed Zn2+-homeostasis gene mRNAs markedly. This might be important because Zn2+ is a major regulator of macrophage activity. Messenger RNAs of seven Zn2+-binding metallothioneins decreased 2–4 fold; the zinc importer SLC39A10 increased 2-fold, in elaidate relative to oleate-treated cells. Results were followed by quantitative PCR comparing cis, trans, and saturated fatty acid effects on Zn2+-homeostasis gene mRNAs. This confirmed that elaidate uniquely decreased metallothionein expression and increased SLC39A10 at 44 hours. Further, intracellular Zn2+ was measured using N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl)amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-phenoxy]-ethoxy]-4-methoxyphenyl]glycine, acetoxymethyl ester (FluoZin-3-AM). This showed that, at 44 hours, only cells treated with elaidate had increased Zn2+. The durable effect of elaidate on Zn2+ activation is a novel and specific effect of trans fatty acids on peripheral macrophage metabolism. PMID:25358453

  11. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  12. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  13. Intracellular Self-Assembly of Cyclic d-Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase.

    PubMed

    Yuan, Yue; Wang, Fuqiang; Tang, Wei; Ding, Zhanling; Wang, Lin; Liang, Lili; Zheng, Zhen; Zhang, Huafeng; Liang, Gaolin

    2016-07-26

    Fatty acid amide hydrolase (FAAH) overexpression induces several disorder symptoms in nerve systems, and therefore long-term tracing of FAAH activity in vivo is of high importance but remains challenging. Current bioluminescence (BL) methods are limited in detecting FAAH activity within 5 h. Herein, by rational design of a latent BL probe (d-Cys-Lys-CBT)2 (1), we developed a "smart" method of intracellular reduction-controlled self-assembly and FAAH-directed disassembly of its cyclic d-luciferin-based nanoparticles (i.e., 1-NPs) for persistent BL imaging of FAAH activity in vitro, in cells, and in vivo. Using aminoluciferin methyl amide (AMA), Lys-amino-d-luciferin (Lys-Luc), and amino-d-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors was controlled by the activity of intracellular FAAH. With the property of long-term tracing of FAAH activity in vivo of 1, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future. PMID:27348334

  14. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  15. Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

    PubMed

    Bellotti, Marta; Salis, Annalisa; Grozio, Alessia; Damonte, Gianluca; Vigliarolo, Tiziana; Galatini, Andrea; Zocchi, Elena; Benatti, Umberto; Millo, Enrico

    2015-01-01

    The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds. PMID:25496807

  16. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  17. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  18. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

    PubMed

    Skatchkov, Serguei N; Bukauskas, Feliksas F; Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V

    2015-06-17

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  19. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels

    PubMed Central

    Skatchkov, Serguei N.; Bukauskas, Feliksas F.; Benedikt, Jan; Inyushin, Mikhail

    2015-01-01

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell–cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  20. Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

    PubMed Central

    Minnigan, H; Moyer, R W

    1985-01-01

    The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses. Images PMID:2991586

  1. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    PubMed

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-01

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  2. Fatty Acid-binding Proteins (FABPs) Are Intracellular Carriers for Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)*

    PubMed Central

    Elmes, Matthew W.; Kaczocha, Martin; Berger, William T.; Leung, KwanNok; Ralph, Brian P.; Wang, Liqun; Sweeney, Joseph M.; Miyauchi, Jeremy T.; Tsirka, Stella E.; Ojima, Iwao; Deutsch, Dale G.

    2015-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  3. Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

    PubMed

    Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

    2016-08-01

    Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. PMID:27209260

  4. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  5. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    PubMed

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  6. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

    PubMed

    Price, Daniel R G; Feng, Honglin; Baker, James D; Bavan, Selvan; Luetje, Charles W; Wilson, Alex C C

    2014-01-01

    Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses. PMID:24367072

  7. RAB24 facilitates clearance of autophagic compartments during basal conditions

    PubMed Central

    Ylä-Anttila, Päivi; Mikkonen, Elisa; Happonen, Kaisa E; Holland, Petter; Ueno, Takashi; Simonsen, Anne; Eskelinen, Eeva-Liisa

    2015-01-01

    RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions. PMID:26325487

  8. The Recycling Endosome of Madin-Darby Canine Kidney Cells Is a Mildly Acidic Compartment Rich in Raft Components

    PubMed Central

    Gagescu, Raluca; Demaurex, Nicolas; Parton, Robert G.; Hunziker, Walter; Huber, Lukas A.; Gruenberg, Jean

    2000-01-01

    We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway. PMID:10930469

  9. Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

    PubMed Central

    Medeiros, Lia Carolina Soares; Gomes, Fabio; Maciel, Luis Renato Maia; Seabra, Sergio Henrique; Docampo, Roberto; Moreno, Silvia; Plattner, Helmut; Hentschel, Joachim; Kawazoe, Urara; Barrabin, Hector; de Souza, Wanderley; DaMatta, Renato Augusto; Miranda, Kildare

    2012-01-01

    The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites. PMID:21699625

  10. Intracellular potassium as a possible inducer of amino acid transport across hamster jejunal enterocytes.

    PubMed Central

    Cremaschi, D; James, P S; Meyer, G; Rossetti, C; Smith, M W

    1986-01-01

    Brush border membrane potentials (Vm), intracellular K+ activity (aiK) and alanine uptake were measured in different parts of villi in mid-jejunal tissue taken from hamsters fed different amounts of food at high and low environmental temperatures. Basal villus enterocytes (Y cells) were found to have lower values for Vm and aiK than upper villus enterocytes (O cells). Alanine uptake was confined to O cells. The position on the villus where values for Vm and aiK changed, and where alanine uptake could first be seen to take place, depended on the energy intake and environmental temperature at which hamsters were kept. Na+-dependent alanine uptake and Vm were both higher in O cells of hamsters fed 10 kcal day-1 at 30 degrees C (10 k/30 degrees C) compared with animals fed 30 kcal day-1 at an environmental temperature of 12 degrees C (30 k/12 degrees C hamsters). The rates at which enterocytes migrated along the crypt-villus axis, measured separately in thymidine-labelling experiments, were 6.9 and 16.1 micron h-1 for 10 k/30 degrees C and 30 k/12 degrees C hamsters respectively. Both Vm and aiK were estimated, from these measurements, to have increased significantly by the time enterocytes became 30 h old. Alanine uptake began 15 h later. Neither of these parameters were influenced by previous adaptation conditions. The close temporal and variable positional relationship found between changes in aiK and onset of transport suggests that early changes in electrolyte composition might initiate a second phase of development enabling the enterocyte to absorb nutrients. The possibility that other ions besides K+ might also be involved in this aspect of regulation is also discussed. PMID:3795055

  11. Acidic amino acids in the first intracellular loop contribute to voltage- and calcium- dependent gating of anoctamin1/TMEM16A.

    PubMed

    Xiao, Qinghuan; Cui, Yuanyuan

    2014-01-01

    Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1. PMID:24901998

  12. Acidic intracellular Ca(2+) stores and caveolae in Ca(2+) signaling and diabetes.

    PubMed

    Guerrero-Hernandez, Agustin; Gallegos-Gomez, Martin Leonardo; Sanchez-Vazquez, Victor Hugo; Lopez-Mendez, Maria Cristina

    2014-11-01

    Acidic Ca(2+) stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca(2+) signaling and we are at the verge of understanding how lysosomes accumulate Ca(2+) and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca(2+) and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes. PMID:25182518

  13. Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

    PubMed

    Young, Kaylie L; Scott, Alexander W; Hao, Liangliang; Mirkin, Sarah E; Liu, Guoliang; Mirkin, Chad A

    2012-07-11

    Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach. PMID:22725653

  14. Listeria monocytogenes varies among strains to maintain intracellular pH homeostasis under stresses by different acids as analyzed by a high-throughput microplate-based fluorometry

    PubMed Central

    Cheng, Changyong; Yang, Yongchun; Dong, Zhimei; Wang, Xiaowen; Fang, Chun; Yang, Menghua; Sun, Jing; Xiao, Liya; Fang, Weihuan; Song, Houhui

    2015-01-01

    Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to determine pHi of listerial cells that were prelabeled with the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester and subjected to acid stress. We found that L. monocytogenes responds differently among strains toward organic and inorganic acids to maintain pHi homeostasis. The capacity of L. monocytogenes to maintain pHi at extracellular pH 4.5 (pHex) was compromised in the presence of acetic acid and lactic acid, but not by hydrochloric acid and citric acid. Organic acids exhibited more inhibitory effects than hydrochloric acid at certain pH conditions. Furthermore, the virulent stains L. monocytogenes EGDe, 850658 and 10403S was more resistant to acidic stress than the avirulent M7 which showed a defect in maintaining pHi homeostasis. Deletion of sigB, a stress-responsive alternative sigma factor from 10403S, markedly altered intracellular pHi homeostasis, and showed a significant growth and survival defect under acidic conditions. Thus, this work provides new insights into bacterial survival mechanism to acidic stresses. PMID:25667585

  15. A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

    PubMed

    Ludwig-Müller, Jutta; Jülke, Sabine; Geiß, Kathleen; Richter, Franziska; Mithöfer, Axel; Šola, Ivana; Rusak, Gordana; Keenan, Sandi; Bulman, Simon

    2015-05-01

    The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids. PMID:25135243

  16. Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

    PubMed Central

    Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

    2013-01-01

    Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

  17. Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

    PubMed

    Yang, Yanfang; Xia, Xuejun; Dong, Wujun; Wang, Hongliang; Li, Lin; Ma, Panpan; Sheng, Wei; Xu, Xueqing; Liu, Yuling

    2016-05-01

    An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. PMID:26822264

  18. Compartmented electrode structure

    DOEpatents

    Vissers, Donald R.; Shimotake, Hiroshi; Gay, Eddie C.; Martino, Fredric J.

    1977-06-14

    Electrodes for secondary electrochemical cells are provided with compartments for containing particles of the electrode reactant. The compartments are defined by partitions that are generally impenetrable to the particles of reactant and, in some instances, to the liquid electrolyte used in the cell. During cycling of the cell, reactant material initially loaded into a particular compartment is prevented from migrating and concentrating within the lower portion of the electrode or those portions of the electrode that exhibit reduced electrical resistance.

  19. Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes.

    PubMed Central

    Livingstone, C; James, D E; Rice, J E; Hanpeter, D; Gould, G W

    1996-01-01

    increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4. PMID:8615819

  20. Compartment Syndrome in Children.

    PubMed

    Hosseinzadeh, Pooya; Hayes, Christopher B

    2016-07-01

    Compartment syndrome in children can present differently than adults. Increased analgesic need should be considered the first sign of evolving compartment syndrome in children. Children with supracondylar humerus fractures, floating elbow injuries, operatively treated forearm fractures, and tibia fractures are at high risk for developing compartment syndrome. Elbow flexion beyond 90° in supracondylar humerus fractures and closed treatment of forearm fractures in floating elbow injuries are associated with increased risk of compartment syndrome. Prompt diagnosis and treatment with fasciotomy in children result in excellent long-term outcomes. PMID:27241380

  1. Preparation, Characterization and Intracellular Imaging of 2,4-Dichlorophenoxyacetic Acid Conjugated Gold Nanorods.

    PubMed

    Jia, Jin-Liang; Jin, Xiao-Yong; Liu, Qing-Le; Liang, Wen-Long; Lin, Miao-Shan; Xu, Han-Hong

    2016-05-01

    Visualizing the biodistribution of pesticides inside living cells is great importance for enhancing targeting of pesticides. Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm x 11.3 nm could be used as a fluorescent tracer to examine the distribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2) cells. The nanostructures of hybrid materials were analyzed by using Raman spectra and X-ray photoelectron spectroscopy (XPS), including spectra assignments and electronic property. These data revealed 2,4-D has successfully conjugated MP-Au NRs according to Raman and XPS. The biodistribution of the conjugates inside BY-2 cells was directly examined at 12 and 24 h by the two-photon microscopy. The intensity of two-photon luminescence (TPL) inside cells demonstrated that the conjugates could be localized and excluded by BY-2 cells. Thus, this labeling approach opens up new avenues to the facile and efficient labeling of pesticides. PMID:27483849

  2. Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

    PubMed

    Franke, Lukáš; Čožíková, Dagmar; Smirnou, Dzianis; Hermannová, Martina; Hanová, Tereza; Růžičková, Andrea; Velebný, Vladimír

    2015-08-01

    Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. PMID:26114654

  3. Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes*

    PubMed Central

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, KeWei

    2012-01-01

    α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders. PMID:22679014

  4. Internalized compartments encapsulated nanogels for targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Yu, Jicheng; Zhang, Yuqi; Sun, Wujin; Wang, Chao; Ranson, Davis; Ye, Yanqi; Weng, Yuyan; Gu, Zhen

    2016-04-01

    Drug delivery systems inspired by natural particulates hold great promise for targeted cancer therapy. An endosome formed by internalization of plasma membrane has a massive amount of membrane proteins and receptors on the surface, which is able to specifically target the homotypic cells. Herein, we describe a simple method to fabricate an internalized compartments encapsulated nanogel with endosome membrane components (EM-NG) from source cancer cells. Following intracellular uptake of methacrylated hyaluronic acid (m-HA) adsorbed SiO2/Fe3O4 nanoparticles encapsulating a crosslinker and a photoinitiator, EM-NG was readily prepared through in situ crosslinking initiated under UV irradiation after internalization. The resulting nanogels loaded with doxorubicin (DOX) displayed enhanced internalization efficiency to the source cells through a specific homotypic affinity in vitro. However, when treated with the non-source cells, the EM-NGs exhibited insignificant difference in therapeutic efficiency compared to a bare HA nanogel with DOX. This study illustrates the potential of utilizing an internalized compartments encapsulated formulation for targeted cancer therapy, and offers guidelines for developing a natural particulate-inspired drug delivery system.Drug delivery systems inspired by natural particulates hold great promise for targeted cancer therapy. An endosome formed by internalization of plasma membrane has a massive amount of membrane proteins and receptors on the surface, which is able to specifically target the homotypic cells. Herein, we describe a simple method to fabricate an internalized compartments encapsulated nanogel with endosome membrane components (EM-NG) from source cancer cells. Following intracellular uptake of methacrylated hyaluronic acid (m-HA) adsorbed SiO2/Fe3O4 nanoparticles encapsulating a crosslinker and a photoinitiator, EM-NG was readily prepared through in situ crosslinking initiated under UV irradiation after internalization. The

  5. Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

    PubMed

    Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

    2016-07-01

    The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. PMID:27091863

  6. Mutations of amino acids in the DNA-recognition domain of Epstein-Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

    SciTech Connect

    Park, Richard; Heston, Lee; Shedd, Duane; Delecluse, Henri-Jacques; Miller, George

    2008-12-20

    ZEBRA, a transcription factor and DNA replication protein encoded by the Epstein-Barr virus (EBV) BZLF1 gene, plays indispensable roles in the EBV lytic cycle. We recently described the phenotypes of 46 single amino acid substitutions introduced into the DNA-recognition region of ZEBRA [Heston, L., El-Guindy, A., Countryman, J., Dela Cruz, C., Delecluse, H.J., and Miller, G. 2006]. The 27 DNA-binding-proficient mutants exhibited distinct defects in their ability to activate expression of the kinetic classes of viral genes. Four phenotypic variants could be discerned: wild-type, defective at activating Rta, defective at activating early genes, and defective at activating late genes. Here we analyze the distribution of ZEBRA within the nucleus and the localization of EA-D (the viral DNA polymerase processivity factor), an indicator of the development of replication compartments, in representatives of each phenotypic group. Plasmids encoding wild-type (WT) and mutant ZEBRA were transfected into 293 cells containing EBV-bacmids. WT ZEBRA protein was diffusely and smoothly distributed throughout the nucleus, sparing nucleoli, and partially recruited to globular replication compartments. EA-D induced by WT ZEBRA was present diffusely in some cells and concentrated in globular replication compartments in other cells. The distribution of ZEBRA and EA-D proteins was identical to WT following transfection of K188R, a mutant with a conservative change. The distribution of S186A mutant ZEBRA protein, defective for activation of Rta and EA-D, was identical to WT, except that the mutant ZEBRA was never found in globular compartments. Co-expression of Rta with S186A mutant rescued diffuse EA-D but not globular replication compartments. The most striking observation was that several mutant ZEBRA proteins defective in activating EA-D (R179A, K181A and A185V) and defective in activating lytic viral DNA replication and late genes (Y180E and K188A) were localized to numerous punctate

  7. Single compartment drug delivery

    PubMed Central

    Cima, Michael J.; Lee, Heejin; Daniel, Karen; Tanenbaum, Laura M.; Mantzavinou, Aikaterini; Spencer, Kevin C.; Ong, Qunya; Sy, Jay C.; Santini, John; Schoellhammer, Carl M.; Blankschtein, Daniel; Langer, Robert S.

    2014-01-01

    Drug design is built on the concept that key molecular targets of disease are isolated in the diseased tissue. Systemic drug administration would be sufficient for targeting in such a case. It is, however, common for enzymes or receptors that are integral to disease to be structurally similar or identical to those that play important biological roles in normal tissues of the body. Additionally, systemic administration may not lead to local drug concentrations high enough to yield disease modification because of rapid systemic metabolism or lack of sufficient partitioning into the diseased tissue compartment. This review focuses on drug delivery methods that physically target drugs to individual compartments of the body. Compartments such as the bladder, peritoneum, brain, eye and skin are often sites of disease and can sometimes be viewed as “privileged,” since they intrinsically hinder partitioning of systemically administered agents. These compartments have become the focus of a wide array of procedures and devices for direct administration of drugs. We discuss the rationale behind single compartment drug delivery for each of these compartments, and give an overview of examples at different development stages, from the lab bench to phase III clinical trials to clinical practice. We approach single compartment drug delivery from both a translational and a technological perspective. PMID:24798478

  8. 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.

  9. Comparing intracellular stability and targeting of sulfobetaine quantum dots with other surface chemistries in live cells.

    PubMed

    Muro, Eleonora; Fragola, Alexandra; Pons, Thomas; Lequeux, Nicolas; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit

    2012-04-10

    The in vivo labeling of intracellular components with quantum dots (QDs) is very limited because of QD aggregation in the cell cytoplasm and/or QD confinement into lysosomal compartments. In order to improve intracellular targeting with QDs, various surface chemistries and delivery methods have been explored, but they have not yet been compared systematically with respect to the QD intracellular stability. In this work, the intracellular aggregation kinetics of QDs for three different surface chemistries based on ligand exchange or encapsulation with amphiphilic polymers are compared. For each surface chemistry, three delivery methods for bringing the nanoparticles into the cells are compared: electroporation, microinjection, and pinocytosis. It is concluded that the QD intracellular aggregation behavior is strongly dependent on the surface chemistry. QDs coated with dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands diffuse freely in cells for longer periods of time than for QDs in the other chemistries tested, and they can access all cytoplasmic compartments. Even when conjugated to streptavidin, these DHLA-SB QDs remain freely diffusing inside the cytoplasm and unaggregated, and they are able to reach a biotinylated target inside HeLa cells. Such labeling was more efficient when compared to commercial streptavidin-conjugated QDs, which may be due to the smaller size of DHLA-SB QDs and/or to their superior intracellular stability. PMID:22378567

  10. Effect of the Fructus Ligustri Lucidi extract and its monomers quercetin and oleanolic acid on the adhesion and migration of melanocytes and intracellular actin

    PubMed Central

    WU, YANHUA; LI, QILIN; LI, XIANGJUN; HE, DANHUA; NIU, MU; LU, XIAOJUAN; LI, HUI

    2016-01-01

    The present study aimed to investigate the effects of the Fructus Ligustri Lucidi (FLL) extract and its monomers quercetin and oleanolic acid on the adhesion and migration of human epidermal melanocytes (MCs) and intracellular actin. The human epidermal MCs were cultured and identified. The cells were treated with different concentrations of FLL extract, quercetin and oleanolic acid. The adhesion and migration abilities of the cells were determined by the fibronectin-coated culture experiment and Transwell assay, respectively. The structure and distribution of intracellular actin were observed by confocal laser microscopy, with semi-quantitative analysis. Results showed that compared with the control group, 0.0375–0.3 mg/ml of the FLL extract and 40 µM quercetin significantly improved the adhesion rate of MCs (P<0.05). The numbers of MCs permeating the microporous membrane in the 0.15 mg/ml FLL extract and 12 µM oleanolic acid groups were 43.7 and 30.3, respectively, significantly higher compared to the control group (P<0.01). In the control group, the intracellular actin was less, and the stress fiber structure was not clear. In the 0.15 mg/ml FLL extract, 12 µM oleanolic acid and 40 µM quercetin groups, there were numerous bunched stress fibers, indicating the aggregation of filamentous fibrous actin. The mean optical densities of actin expression in the 0.15 mg/ml FLL extract, 12 µM oleanolic acid and 40 µM quercetin groups were significantly higher compared to the control group (P<0.05). The FLL extract has a significant stimulatory effect on the adhesion and migration of human epidermal MCs. The mechanism may be associated with the promotion of intracellular actin cytoskeleton aggregation. PMID:27123251

  11. Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging.

    PubMed

    Manunta, Maria; Izzo, Lorella; Duncan, Ruth; Jones, Arwyn Tomos

    2007-01-01

    As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep) density gradient optimised to 5-20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-beta-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation

  12. Chronic Exertional Compartment Syndrome.

    PubMed

    Braver, Richard T

    2016-04-01

    Increased tissue pressure within a fascial compartment may be the result from any increase in volume within its contents, or any decrease in size of the fascial covering or its distensibility. This may lead to symptoms of leg tightness, pain or numbness brought about by exercise. There are multiple differential diagnoses of exercise induced leg pain and the proper diagnoses of chronic exertional compartment syndrome (CECS) is made by a careful history and by exclusion of other maladies and confirmed by compartment syndrome testing as detailed in this text. Surgical fasciotomies for the anterior, lateral, superficial and deep posterior compartments are described in detail along with ancillary procedures for chronic shin splints that should allow the athlete to return to competitive activity. PMID:27013413

  13. Chronic exertional compartment syndrome.

    PubMed

    George, Christopher A; Hutchinson, Mark R

    2012-04-01

    Chronic exertional compartment syndrome is a relatively common, but often overlooked cause of leg pain in athletes. A careful history and physical examination is essential in the diagnosis of CECS. Affected individuals have recurrent, activity-related leg pain that recurs at a consistent duration or intensity and is only relieved by rest. Measurement of baseline and postexercise compartment pressures confirms the diagnosis and helps in the planning of treatment. Surgical treatment with fasciotomy of the involved compartments is successful in allowing patients to return to full activity levels. With surgical treatment, it is critical to address all affected compartments as well as releasing any fascial defects, both of which may cause recurrent symptoms if neglected. With appropriate diagnosis and treatment, excellent outcomes can be achieved and allow athletes to return to full, unrestricted activity levels. PMID:22341019

  14. Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane

    SciTech Connect

    Ray, R.; Legere, R.H.; Majerus, B.J.; Petrali, J.P.

    1995-12-31

    The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in thai vitro mouse neuroblastoma-rat glioma hybrid NG 108-1 S clonal p cell line model. Following 0.3 mM SM exposure, cell viability remained high (>80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover,there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ((3H)AA) from cellular membranes. Increase in (3H)AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in I3HIAA release was inhibited by quinacrine (20 uM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in I3HIAA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca2+-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

  15. Abdominal Compartment Hypertension and Abdominal Compartment Syndrome.

    PubMed

    Maluso, Patrick; Olson, Jody; Sarani, Babak

    2016-04-01

    Intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) are rare but potentially morbid diagnoses. Clinical index of suspicion for these disorders should be raised following massive resuscitation, abdominal wall reconstruction/injury, and in those with space-occupying disorders in the abdomen. Gold standard for diagnosis involves measurement of bladder pressure, with a pressure greater than 12 mm Hg being consistent with IAH and greater than 25 mm Hg being consistent with ACS. Decompressive laparotomy is definitive therapy but paracentesis can be equally therapeutic in properly selected patients. Left untreated, ACS can lead to multisystem organ failure and death. PMID:27016163

  16. Cluster of Differentiation 38 (CD38) Mediates Bile Acid-induced Acinar Cell Injury and Pancreatitis through Cyclic ADP-ribose and Intracellular Calcium Release*

    PubMed Central

    Orabi, Abrahim I.; Muili, Kamaldeen A.; Javed, Tanveer A.; Jin, Shunqian; Jayaraman, Thottala; Lund, Frances E.; Husain, Sohail Z.

    2013-01-01

    Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca2+ signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38−/−). Cytosolic Ca2+ signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μm). To focus on intracellular Ca2+ release and to specifically exclude Ca2+ influx, cells were perifused in Ca2+-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mm) or the cADPR antagonist 8-Br-cADPR (30 μm) abrogated TLCS-induced Ca2+ signals and cell injury. TLCS-induced Ca2+ release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca2+ signaling. PMID:23940051

  17. Spacecraft Compartment Venting

    NASA Technical Reports Server (NTRS)

    Scialdone, John J.

    1998-01-01

    At various time concerns have been expressed that rapid decompressions of compartments of gas pockets and thermal blankets during spacecraft launches may have caused pressure differentials across their walls sufficient to cause minor structural failures, separations of adhesively-joined parts, ballooning, and flapping of blankets. This paper presents a close form equation expressing the expected pressure differentials across the walls of a compartment as a function of the external to the volume pressure drops, the pressure at which the rates occur and the vent capability of the compartment. The pressure profiles measured inside the shrouds of several spacecraft propelled by several vehicles and some profiles obtained from ground vacuum systems have been included. The equation can be used to design the appropriate vent, which will preclude excessive pressure differentials. Precautions and needed approaches for the evaluations of the expected pressures have been indicated. Methods to make a rapid assessment of the response of the compartment to rapid external pressure drops have been discussed. These are based on the evaluation of the compartment vent flow conductance, the volume and the length of time during which the rapid pressure drop occurs.

  18. The intracellular parasite Toxoplasma gondii depends on the synthesis of long chain and very long-chain unsaturated fatty acids not supplied by the host cell

    PubMed Central

    Ramakrishnan, Srinivasan; Docampo, Melissa D.; MacRae, James I.; Ralton, Julie E.; Rupasinghe, Thusitha; McConville, Malcolm J.; Striepen, Boris

    2015-01-01

    SUMMARY Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In T. gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here we demonstrate that the ER-localized fatty acid elongation (ELO) is essential for parasite growth. Conditional knock-down of the non-redundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. 13C-glucose and 13C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was by-passed by supplementing the media with specific fatty acids, indicating active, but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway. PMID:25825226

  19. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  20. Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE.

    PubMed

    Liu, Yingbao; Yang, Xiaobing; Yin, Yajie; Lin, Jinshui; Chen, Can; Pan, Junfeng; Si, Meiru; Shen, Xihui

    2016-07-14

    Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass. PMID:27250661

  1. Application of a combined effect compartment and binding model for gastric acid inhibition of AR-HO47108: a potassium competitive acid blocker, and its active metabolite AR-HO47116 in the dog.

    PubMed

    Abelö, Angela; Andersson, Magdalena; Holmberg, Ann Aurell; Karlsson, Mats O

    2006-10-01

    The effect of AR-HO47108, a potassium competitive acid blocker, and its active metabolite AR-HO47116 was studied in Heidenhain pouch dogs following administration of single oral and intravenous doses of the two compounds. The histamine-stimulated acid secretion was measured in different periods after dose up to 24h. All data obtained in the different studies was pooled and analyzed by non-linear mixed effects modelling. It was found that there is a delay between the plasma concentration-time peak and the maximum inhibitory effect and that the effect persisted longer than anticipated from the plasma concentration half-lives of the compounds. In addition, it was found that the peak effect was reached earlier at higher doses. The effect data was well described by a combined effect compartment and binding model and both distribution to the biophase i.e. the canaliculus of the parietal cell and a rate limiting binding interaction between drug and enzyme appear to contribute to the observed delay. In addition, a secretion rate dependent washout from the biophase may contribute. Furthermore, because the parent compound and metabolite bind to the same enzyme, the effect is determined by competition between the two for the same enzyme. The metabolite was found to be less potent than the parent compound, with Kd values of the combined model of 125 and 11.2 nM for the metabolite and parent compound, respectively. However, the metabolite is generated in high concentrations that rapidly exceed the concentration of parent compound after oral administration of parent compound, and this together with its longer plasma half-life will make its contribution to the overall effect increase with time and slightly prolong the duration of the effect. PMID:16831536

  2. The Crew Compartment Trainer

    NASA Technical Reports Server (NTRS)

    1998-01-01

    STS-93 crew emergency egress training in the Crew Compartment Trainer (CCT). The five crewmembers of STS-93 in the middeck mock-up are from left to right: Mission Specialist Michel Tognini, Mission Specialist Catherine 'Cady' Coleman, Pilot Jeffrey Ashby, Commander Eileen Collins and Mission Specialist Stephen Hawley.

  3. [Chronic exertional compartment syndrome].

    PubMed

    Rom, Eyal; Tenenbaum, Shay; Chechick, Ofir; Burstein, Gideon; Amit, Yehuda; Thein, Ran

    2013-10-01

    Chronic exertional compartment syndrome is an uncommon phenomenon first reported in the mid 50's. This condition is characterized by sharp pain during physical activity, causing reduction in activity frequency or intensity and even abstention. This syndrome is caused by elevation of the intra-compartmental pressure which leads to decreased tissue perfusion, thus ischemic damage to the tissue ensues. Chronic exertional syndrome is usually related to repetitive physical activity, usually in young people and athletes. The physical activity performed by the patient causes a rise in intra-compartmental pressure and thereby causes pain. The patient discontinues the activity and the pain subsides within minutes of rest. Chronic exertional syndrome is reported to occur in the thigh, shoulder, arm, hand, foot and gluteal region, but most commonly in the leg, especially the anterior compartment. The diagnosis of chronic exertional syndrome is primarily based on patients' medical history, supported by intramuscular pressure measurement of the specific compartment involved. Treatment of chronic exertional syndrome, especially the anterior and lateral compartment of the leg is mainly by surgery i.e. fasciotomy. If the patient is reluctant to undergo a surgical procedure, the conservative treatment is based on abstention from the offending activity, changing footwear or using arch support. However, the conservative approach is not as successful as surgical treatment. PMID:24450036

  4. Activation of 5-( sup 125 I)iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

    SciTech Connect

    Rosenwald, A.G.; Pagano, R.E.; Raviv, Y. )

    1991-05-25

    We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-({sup 125}I)iodonaphthyl-1-azide (({sup 125}I)INA). ({sup 125}I) INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with {sup 125}I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for ({sup 125}I)INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with {sup 125}I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of ({sup 125}I)INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.

  5. Leelamine mediates cancer cell death through inhibition of intracellular cholesterol transport.

    PubMed

    Kuzu, Omer F; Gowda, Raghavendra; Sharma, Arati; Robertson, Gavin P

    2014-07-01

    Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray-based analyses of the intracellular signaling cascades showed alterations in RTK-AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers. PMID:24688051

  6. Leelamine mediates cancer cell death through inhibition of intracellular cholesterol transport

    PubMed Central

    Kuzu, Omer F.; Gowda, Raghavendra; Sharma, Arati; Robertson, Gavin P.

    2015-01-01

    Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray–based analyses of the intracellular signaling cascades showed alterations in RTK–AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers. PMID:24688051

  7. 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

  8. 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

  9. Computer model of unstirred layer and intracellular pH changes. Determinants of unstirred layer pH.

    PubMed

    Marrannes, Roger

    2013-06-01

    Transmembrane acid-base fluxes affect the intracellular pH and unstirred layer pH around a superfused biological preparation. In this paper the factors influencing the unstirred layer pH and its gradient are studied. An analytical expression of the unstirred layer pH gradient in steady state is derived as a function of simultaneous transmembrane fluxes of (weak) acids and bases with the dehydration reaction of carbonic acid in equilibrium. Also a multicompartment computer model is described consisting of the extracellular bulk compartment, different unstirred layer compartments and the intracellular compartment. With this model also transient changes and the influence of carbonic anhydrase (CA) can be studied. The analytical expression and simulations with the multicompartment model demonstrate that in steady state the unstirred layer pH and its gradient are influenced by the size and type of transmembrane flux of acids and bases, their dissociation constant and diffusion coefficient, the concentration, diffusion coefficient and type of mobile buffers and the activity and location of CA. Similar principles contribute to the amplitude of the unstirred layer pH transients. According to these models an immobile buffer does not influence the steady-state pH, but reduces the amplitude of pH transients especially when these are fast. The unstirred layer pH provides useful information about transmembrane acid-base fluxes. This paper gives more insight how the unstirred layer pH and its transients can be interpreted. Methodological issues are discussed. PMID:23860924

  10. Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

    SciTech Connect

    Russell, J.B. )

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y{sub ATP} (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up ({sup 14}C)acetate and ({sup 14}C)benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.

  11. Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation.

    PubMed Central

    Russell, J B

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation. PMID:2036013

  12. Depollution potential of three macrophytes: exudated, wall-bound and intracellular peroxidase activities plus intracellular phenol concentrations.

    PubMed

    Larue, Camille; Korboulewsky, Nathalie; Wang, Runying; Mévy, Jean-Philippe

    2010-10-01

    The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 3.58 mg eq. [corrected] gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals. PMID:20570142

  13. Acute Compartment Syndrome.

    PubMed

    Schmidt, Andrew H

    2016-07-01

    Acute compartment syndrome (ACS) is a well-known pathophysiologic complication of trauma or tissue ischemia. ACS affects the appearance, function, and even the viability of the involved limb, and demands immediate diagnosis and treatment. However, ACS is difficult to diagnose and the only effective treatment is decompressive surgical fasciotomy. The clinical signs and symptoms may easily be attributed to other aspects of the injury, which further complicates the diagnosis. This article highlights the latest information regarding the diagnosis of ACS, how to perform fasciotomies, how to manage fasciotomy wounds, and also reviews complications and outcomes of ACS. PMID:27241376

  14. 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

  15. 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

  16. Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments.

    PubMed

    Brumell, J H; Tang, P; Mills, S D; Finlay, B B

    2001-09-01

    Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event. PMID:11555418

  17. Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

    PubMed Central

    Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

    2013-01-01

    N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

  18. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier.

    PubMed

    Geng, Yao; Lin, Dajie; Shao, Lijia; Yan, Feng; Ju, Huangxian

    2013-01-01

    A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics. PMID:23762388

  19. Intracellular calcium-release and protein kinase C-activation stimulate sonic hedgehog gene expression during gastric acid secretion

    PubMed Central

    El-Zaatari, Mohamad; Zavros, Yana; Tessier, Art; Waghray, Meghna; Lentz, Steve; Gumucio, Deborah; Todisco, Andrea; Merchant, Juanita L.

    2010-01-01

    Introduction Hypochlorhydria during Helicobacter pylori infection inhibits gastric Shh expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through Ca2+i-dependent protein kinase C (PKC) or cAMP-dependent protein kinase A (PKA)-activation. Method We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1 (sHip-1), a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, EGTA+BAPTA, PKC-overexpressing adenoviruses, and PKC-inhibitors were used to modulate Ca2+i-release, PKC-activity and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H+/K+-β-cholera-toxin overexpressing mice (Ctox). Results Mice that expressed sHip-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression was also repressed in the hyperchlorhydric Ctox model with elevated cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca2+i-release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin- and carbachol-mediated release of Ca2+i induced Shh expression. Ca2+-chelation with BAPTA+EGTA reduced Shh expression. Overexpression of PKC-α, -β and -δ (but not PKC-ε) induced Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene. Conclusion Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca2+i-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin. PMID:20816837

  20. Administration of Exogenous Growth Hormone Is Associated with Changes in Plasma and Intracellular Mammary Amino Acid Profiles and Abundance of the Mammary Gland Amino Acid Transporter SLC3A2 in Mid-Lactation Dairy Cows

    PubMed Central

    Sciascia, Quentin L.; Pacheco, David; McCoard, Susan A.

    2015-01-01

    The objectives of this study were to (1) identify changes in plasma and mammary intracellular amino acid (AA) profiles in dairy cows treated with growth hormone (GH), and (2) evaluate the expression of mammary gland genes involved in the transport of AA identified in (1). Eight non-pregnant (n = 4 per group) lactating dairy cows were treated with a single subcutaneous injection of either a slow-release formulation of commercially available GH (Lactotropin 500 mg) or physiological saline solution. Six days after treatment, cows were milked and blood collected from the jugular vein for the analysis of free AA in the plasma. Cows were euthanized and mammary tissue harvested. Treatment with GH increased milk, protein, fat and lactose yields, with no effect on dry matter intake. Plasma concentrations of lysine and group I AA decreased significantly, and arginine, methionine, tyrosine and arginine-family AA tended to decrease in GH-treated cows. Concentrations of intracellular glycine, serine and glutamate increased significantly, with a trend for decreased arginine observed in the mammary gland of GH-treated cows. A trend for increased concentrations of intracellular total AA, NEAA and arginine-family AA were observed in the mammary gland of GH-treated cows. Variance in the concentration of plasma methionine, tyrosine, valine, alanine, ornithine, BCAA, EAA was significantly different between treatments. Variance in the concentration of intracellular lysine, valine, glutamine, EAA and group II was significantly different between treatments. AA changes were associated with increased mRNA abundance of the mammary gland AA transporter SLC3A2. We propose that these changes occur to support increased milk protein and fatty acid production in the mammary gland of GH-treated cows via potential mTOR pathway signaling. PMID:26226162

  1. 5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

    SciTech Connect

    Mi Wei; Li Lanfen; Su Xiaodong

    2008-04-18

    Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys{sup 114}, and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general.

  2. Modulation of spontaneous transmitter release from the frog neuromuscular junction by interacting intracellular Ca(2+) stores: critical role for nicotinic acid-adenine dinucleotide phosphate (NAADP).

    PubMed Central

    Brailoiu, Eugen; Patel, Sandip; Dun, Nae J

    2003-01-01

    Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca(2+)-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca(2+)-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP(3)), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP(3), cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca(2+) stores with distinct Ca(2+) stores targeted by NAADP. PMID:12749764

  3. Intracellular delivery of desulfated heparin with bile acid conjugation alleviates T cell-mediated inflammatory arthritis via inhibition of RhoA-dependent transcellular diapedesis.

    PubMed

    Kang, Jin Hee; Hwang, Seung Rim; Sung, Shijin; Jang, Ji Ae; Alam, Md Mahmudul; Sa, Keum Hee; Kim, Sang-Yeob; Kim, In San; Byun, Young Ro; Kang, Young Mo

    2014-06-10

    Heparin has a potential regulatory role in inflammatory diseases. However, the anticoagulant activity and poor oral bioavailability of heparin limit its use as an anti-inflammatory agent. Conjugation of bis-deoxycholic acid to 6-O-desulfated low molecular weight heparin (6DSHbD) was efficiently internalized by activated endothelial cells via a 2-step model, in which heparin attaches to adhesion molecules that facilitate accessibility of the bile acid conjugate to membrane transporters. The critical role of P-selectin during endothelial cell uptake of 6DSHbD by arthritic tissue was confirmed in p-selectin(-/-) arthritic mice. Intracellular 6DSHbD inhibited transcellular diapedesis of T cells through activated endothelial cells and impaired both the formation of ICAM-1-rich docking structures at the T cell contact surface and subsequent cytoskeletal rearrangement. Furthermore, 6DSHbD blocked activation of RhoA-GTPase and phosphorylation of ezrin/radixin/moesin induced by ICAM-1 cross-linking on activated endothelial cells, thereby impairing lymphocyte transcellular transmigration. After oral administration 6DSHbD was preferentially delivered to inflamed joint tissue, particularly in and around post-capillary venular endothelium and inhibited effector T cell homing to arthritic joints. Aggravation of collagen-induced arthritis conferred by the transfer of effector T cells was suppressed by oral 6DSHbD. Thus, intracellular heparin exerts anti-inflammatory effects through the inhibition of RhoA-dependent transendothelial recruitment of T cells and may have applications in the treatment of chronic inflammatory arthritis. PMID:24657949

  4. Compartment Syndrome of the Hand.

    PubMed

    Oak, Nikhil R; Abrams, Reid A

    2016-07-01

    Hand compartment syndrome has many etiologies; untreated, it has dire functional consequences. Intracompartmental pressure exceeding capillary filling pressure causes decreased tissue perfusion resulting in progressive ischemic death of compartment contents. Clinical findings can evolve. Serial physical examinations are recommended and, if equivocal, interstitial pressure monitoring is indicated. Definitive management is emergent fasciotomies with incisions designed to decompress the involved hand compartments, which could include the thenar, hypothenar, and interosseous compartments, and the carpal tunnel. Careful wound care, edema management, splinting, and hand therapy are critical. Therapy should start early postoperatively, possibly before wound closure. PMID:27241383

  5. 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.

  6. 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

  7. Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni▿ †

    PubMed Central

    Sumby, Krista M.; Matthews, Angela H.; Grbin, Paul R.; Jiranek, Vladimir

    2009-01-01

    We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates. PMID:19734337

  8. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    SciTech Connect

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  9. COMPARTMENTED REACTOR FUEL ELEMENT

    DOEpatents

    Cain, F.M. Jr.

    1962-09-11

    A method of making a nuclear reactor fuel element of the elongated red type is given wherein the fissionable fuel material is enclosed within a tubular metal cladding. The method comprises coating the metal cladding tube on its inside wall with a brazing alloy, inserting groups of cylindrical pellets of fissionable fuel material into the tube with spacing members between adjacent groups of pellets, sealing the ends of the tubes to leave a void space therewithin, heating the tube and its contents to an elevated temperature to melt the brazing alloy and to expand the pellets to their maximum dimensions under predetermined operating conditions thereby automatically positioning the spacing members along the tube, and finally cooling the tube to room temperature whereby the spacing disks become permanently fixed at their edges in the brazing alloy and define a hermetically sealed compartment for each fl group of fuel pellets. Upon cooling, the pellets contract thus leaving a space to accommodate thermal expansion of the pellets when in use in a reactor. The spacing members also provide lateral support for the tubular cladding to prevent collapse thereof when subjected to a reactor environment. (AEC)

  10. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    PubMed Central

    2012-01-01

    Background Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. Results Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. Conclusion Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the

  11. Modification of intracellular free calcium in cultured A10 vascular smooth muscle cells by exogenous phosphatidic acid.

    PubMed

    Bhugra, Praveen; Xu, Yan-Jun; Rathi, Satyajeet; Dhalla, Naranjan S

    2003-06-15

    Exogenous phosphatidic acid (PA) was observed to produce a concentration-dependent increase in [Ca(2+)](i) in cultured A10 vascular smooth muscle cells. Preincubation of cells with sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (cyclopiazonic acid and thapsigargin), a phospholipase C inhibitor (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), inositol 1,4,5-trisphosphate receptor antagonists (2-aminoethoxydiphenyl borate and xestospongin), and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate) depressed the PA-evoked increase in [Ca(2+)](i). Although EGTA, an extracellular Ca(2+) chelator, decreased the PA-induced increase in [Ca(2+)](i), sarcolemmal Ca(2+)-channel blockers (verapamil or diltiazem) did not alter the action of PA. On the other hand, inhibitors of PKC (bisindolylmaleimide I) and G(i)-protein (pertussis toxin) potentiated the increase in [Ca(2+)](i) evoked by PA significantly. These results suggest that the PA-induced increase in [Ca(2+)](i) in vascular smooth muscle cells may occur upon the activation of phospholipase C and the subsequent release of Ca(2+) from the inositol 1,4,5-trisphosphate-sensitive Ca(2+) pool in the sarcoplasmic reticulum. This action of PA may be mediated through the involvement of PKC. PMID:12787890

  12. pH-sensitive micelles based on acid-labile pluronic F68-curcumin conjugates for improved tumor intracellular drug delivery.

    PubMed

    Fang, Xiao-Bin; Zhang, Jin-Ming; Xie, Xi; Liu, Di; He, Cheng-Wei; Wan, Jian-Bo; Chen, Mei-Wan

    2016-04-11

    Curcumin (Cur) is a highly pleiotropic anticancer agent that inhibits cell proliferation and induces apoptosis in cancer cells. A variety of nano-systems constituted by polymer-drug conjugates have been designed to overcome its shortages on water solubility, chemical instability, and poor bioavailability. However, most of them suffer from ineffective release of Cur in cancer cells in vivo. This work developed a novel flexible acid-responsive micelle formulation by covalently conjugating Cur on the hydrophilic terminals of pluronic F68 chains via cis-aconitic anhydride linkers. The synthesized F68-Cis-Cur conjugates can readily precipitate to form homogeneous micelles with average size about 100nm in aqueous solution. In acid environments, F68-Cis-Cur conjugates would break down and subsequently release Cur rapidly, for the reason of pH-sensitive cleavage of cis-aconitic anhydride linkers. In vitro anticancer activity tests demonstrated that F68-Cis-Cur micelles induced higher cytotoxicity against both A2780 and SMMC 7721 cells than free Cur. It provided a larger decrease of mitochondrion membrane potential and induced cellular apoptosis. F68-Cis-Cur micelles remarkably increased cellular uptake of Cur than free Cur through caveolae-mediated endocytosis in an energy-dependent manner. This study demonstrates F68-Cis-Cur conjugation as a promising tool for improving intracellular drug delivery in cancer therapy. PMID:26784981

  13. 3β-Acetyl tormentic acid reverts MRP1/ABCC1 mediated cancer resistance through modulation of intracellular levels of GSH and inhibition of GST activity.

    PubMed

    Rocha, Gleice da Graça; Oliveira, Rodrigo Rodrigues; Kaplan, Maria Auxiliadora Coelho; Gattass, Cerli Rocha

    2014-10-15

    ABC transporter overexpression is an important mechanism of multidrug resistance (MDR) and one of the main obstacles to successful cancer treatment. As these proteins actively remove chemotherapeutics from the tumor cells, the pharmacological inhibition of their activity is a possible strategy to revert drug resistance. Moreover, the ability of MDR inhibitors to sensitize resistant cells to conventional drugs is important for their clinical use. Evidence has shown that the multidrug resistance protein 1 (MRP1/ABCC1) is a negative prognostic marker in patients with lung, gastric, or breast cancers or neuroblastoma. Previous data have shown that 3β-acetyl tormentic acid (3ATA) inhibits the transport activity of the protein MRP1/ABCC1. In this study, we evaluated the ability of 3ATA to sensitize an MDR cell line (GLC4/ADR), which overexpresses MRP1, and investigated the anti-MRP1 mechanisms activated by 3ATA. The results showed that 3ATA is able to reverse the resistance of the MDR cell line to doxorubicin and vincristine, two drugs that are commonly used in cancer chemotherapy. Regarding the sensitizing mechanism induced by 3ATA, this work shows that the triterpene does not modulate the expression of MRP1/ABCC1 but is able to reduce total intracellular glutathione (GSH) levels and decrease the activity of glutathione-s-transferase (GST), the enzyme responsible for the glutathione conjugation of xenobiotics. Together, these results show that 3ATA sensitizes the MDR cell line overexpressing MRP1/ABCC1 to antineoplastic drugs and that this effect is mediated by the modulation of intracellular levels of GSH and GST activity. PMID:25111243

  14. 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

  15. Bioreducible Polycations as Shuttles for Therapeutic Nucleic Acid and Protein Transfection

    PubMed Central

    Klein, Philipp M.

    2014-01-01

    Abstract Significance: Nucleic acids such as gene-encoding DNAs, gene-silencing small interfering RNAs, or recombinant proteins addressing intracellular molecular targets present a major new therapeutic modality, provided efficient solutions for intracellular delivery can be found. The different physiological redox environments inside and outside the cell can be utilized for optimizing the involved transport processes. Recent Advances: Intracellular delivery of nucleic acids or proteins requires dynamic carriers that discriminate between different cellular locations. Bioreducible cationic polymers can package their therapeutic cargo stably in the extracellular environment, but sense the reducing intracellular cytosolic environment. Based on disulfide cleavage, carriers are degraded into biocompatible fragments and release the cargo in functional form. Disulfide linkages between oligocations, between the carrier and the cargo, or spatial caging of complexed cargo by disulfides have been pursued, with polymers or precise sequence-defined peptides and oligomers. Critical Issues: A quantitative knowledge of the bioreductive capacities within different biological compartments and the involved cellular reduction processes would be greatly helpful for improved carriers with disulfides cleaved within the right compartment at the right time. Future Directions: Novel designs of multifunctional nanocarriers will incorporate macromolecular disulfide entry mechanisms previously optimized by natural evolution of toxins and viruses. In addition to extracellular stabilization and intracellular disassembly, tuned disulfides will contribute to deshielding at the cell surface, or translocation from intracellular compartments to the cytosol. Antioxid. Redox Signal. 21, 804–817. PMID:24219092

  16. Curcumin inhibits intracellular fatty acid synthase and induces apoptosis in human breast cancer MDA-MB-231 cells.

    PubMed

    Fan, Huijin; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; Sun, Jia; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-05-01

    High levels of fatty acid synthase (FAS) expression have been found in many tumors, including prostate, breast, and ovarian cancers, and inhibition of FAS has been reported to obstruct tumor growth in vitro and in vivo. Curcumin is one of the major active ingredients of Curcuma longa, which has been proven to inhibit the growth of cancer cells. In the present study, we investigated the potential activity of curcumin as a FAS inhibitor for chemoprevention of breast cancer. As a result, curcumin induced human breast cancer MDA-MB-231 cell apoptosis with the half-inhibitory concentration value of 3.63 ± 0.26 µg/ml, and blocked FAS activity, expression and mRNA level in a dose-dependent manner. Curcumin also regulated B-cell lymphoma 2 (Bcl-2), Bax and p-Akt protein expression in MDA-MB-231 cells. Moreover, FAS knockdown showed similar effect as curcumin. All these results suggested that curcumin may induce cell apoptosis via inhibiting FAS. PMID:26985864

  17. Abscisic acid-induced rearrangement of intracellular structures associated with freezing and desiccation stress tolerance in the liverwort Marchantia polymorpha.

    PubMed

    Akter, Khaleda; Kato, Masahiro; Sato, Yuki; Kaneko, Yasuko; Takezawa, Daisuke

    2014-09-15

    The plant growth regulator abscisic acid (ABA) is known to be involved in triggering responses to various environmental stresses such as freezing and desiccation in angiosperms, but little is known about its role in basal land plants, especially in liverworts, representing the earliest land plant lineage. We show here that survival rate after freezing and desiccation of Marchantia polymorpha gemmalings was increased by pretreatment with ABA in the presence of increasing concentrations of sucrose. ABA treatment increased accumulation of soluble sugars in gemmalings, and sugar accumulation was further increased by addition of sucrose to the culture medium. ABA treatment of gemmalings also induced accumulation of transcripts for proteins with similarity to late embryogenesis abundant (LEA) proteins, which accumulate in association with acquisition of desiccation tolerance in maturing seeds. Observation by light and electron microscopy indicated that the ABA treatment caused fragmentation of vacuoles with increased cytosolic volume, which was more prominent in the presence of a high concentration of external sucrose. ABA treatment also increased the density of chloroplast distribution and remarkably enlarged their volume. These results demonstrate that ABA induces drastic physiological changes in liverwort cells for stress tolerance, accompanied by accumulation of protectants against dehydration and rearrangement and morphological alterations of cellular organelles. PMID:25046754

  18. Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy.

    PubMed

    Gao, Lian-Yong; Laval, Francoise; Lawson, Elise H; Groger, Richard K; Woodruff, Andy; Morisaki, J Hiroshi; Cox, Jeffery S; Daffe, Mamadou; Brown, Eric J

    2003-09-01

    Mycobacterium tuberculosis infects one-third of the world's population and causes two million deaths annually. The unusually low permeability of its cell wall contributes to the ability of M. tuberculosis to grow within host macrophages, a property required for pathogenesis of infection. Mycobacterium marinum is an established model for discovering genes involved in mycobacterial infection. Mycobacterium marinum mutants with transposon insertions in the beta-ketoacyl-acyl carrier protein synthase B gene (kasB) grew poorly in macrophages, although growth in vitro was unaffected. Detailed analyses by thin-layer chromatography, nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, infrared spectroscopy, and chemical degradations showed that the kasB mutants synthesize mycolic acids that are 2-4 carbons shorter than wild type; the defect was localized to the proximal portion of the meromycolate chain. In addition, these mutants showed a significant (approximately 30%) reduction in the abundance of keto-mycolates, with a slight compensatory increase of both alpha- and methoxy-mycolates. Despite these small changes in mycolate length and composition, the kasB mutants exhibited strikingly altered cell wall permeability, leading to a marked increase in susceptibility to lipophilic antibiotics and the host antimicrobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were fully complemented by expressing M. tuberculosis kasB, but not by the closely related gene kasA. These studies identify kasB as a novel target for therapeutic intervention in mycobacterial diseases. PMID:12950920

  19. Development and enteral long-chain n-3 fatty acids differentially alters muscle intracellular pools of free amino acids in the neonate piglet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent studies suggest that feeding long-chain n-3 fatty acids (LCn-3FA) in the diet may blunt the developmental reduction in insulin sensitivity and anabolism in the neonate piglet. To examine the effect of LCn-3FA on protein anabolism, 2-day-old piglets (n=28) were weaned and assigned to one of t...

  20. Compartments of the foot: topographic anatomy.

    PubMed

    Faymonville, C; Andermahr, J; Seidel, U; Müller, L P; Skouras, E; Eysel, P; Stein, G

    2012-12-01

    Recent publications have renewed the debate regarding the number of foot compartments. There is also no consensus regarding allocation of individual muscles and communication between compartments. The current study examines the anatomic topography of the foot compartments anew using 32 injections of epoxy-resin and subsequent sheet plastination in 12 cadaveric foot specimens. Six compartments were identified: dorsal, medial, lateral, superficial central, deep forefoot, and deep hindfoot compartments. Communication was evident between the deep hindfoot compartment and the superficial central and deep central forefoot compartments. In the hindfoot, the neurovascular bundles were located in separate tissue sheaths between the central hindfoot compartment and the medial compartment. In the forefoot, the medial and lateral bundles entered the deep central forefoot compartment. The deep central hindfoot compartment housed the quadratus plantae muscle, and after calcaneus fracture could develop an isolated compartment syndrome. PMID:22638720

  1. Accumulation of intra-cellular polyphosphate in Chlorella vulgaris cells is related to indole-3-acetic acid produced by Azospirillum brasilense.

    PubMed

    Meza, Beatriz; de-Bashan, Luz E; Hernandez, Juan-Pablo; Bashan, Yoav

    2015-06-01

    Accumulation of intra-cellular phosphate, as polyphosphate, was measured when the microalga Chlorella vulgaris was immobilized in alginate with either of two wild-type strains of the microalgae growth-promoting bacterium Azospirillum brasilense or their corresponding IAA-attenuated mutants. Wild type strains of A. brasilense induced higher amounts of intra-cellular phosphate in Chlorella than their respective mutants. Calculations comparing intra-cellular phosphate accumulation by culture or net accumulation by the cell and the amount of IAA that was produced by each of these strains revealed that higher IAA was linked to higher accumulations of intra-cellular phosphate. Application of four levels of exogenous IAA reported for A. brasilense and their IAA-attenuated mutants to cultures of C. vulgaris enhanced accumulation of intra-cellular phosphate; the higher the content of IAA per culture or per single cell, the higher was the amount of accumulated phosphate. When an IAA-attenuated mutant was complemented with exogenous IAA, accumulation of intra-cellular phosphate at the culture level was even higher than phosphate accumulation with the respective wild type strains. When calculating the net accumulation of intra-cellular phosphate in the complementation experiment, net intra-cellular phosphate induced by the IAA-attenuated mutant was completely restored and was similar to the wild strains. We propose that IAA produced by A. brasilense is linked to polyphosphate accumulation in C. vulgaris. PMID:25797155

  2. Endoscopic compartment release for chronic exertional compartment syndrome.

    PubMed

    Knight, Justin R; Daniels, Marissa; Robertson, William

    2013-05-01

    Exertional compartment syndrome of the leg is a condition that can cause chronic debilitating pain in active persons during a variety of aerobic activities. Nonoperative treatments using stretching protocols and activity modifications are often unsuccessful, and thus several operative strategies have been used to treat this condition. A novel technique for endoscopically assisted fasciotomy for chronic exertional compartment syndrome is described. By use of a small laterally based incision and an arthroscope, polydioxanone sutures are passed percutaneously along the anterior and lateral compartments with the Spectrum suture-shuttling device (ConMed Linvatec, Largo, FL). These sutures are used to retract the skin and subcutaneous tissues over the respective compartments. This method allows excellent visualization of the intercompartmental septum, the superficial peroneal nerve, and all perforating vessels. The anterior and lateral compartments can be safely and completely released with this minimally invasive approach. The patient is allowed to return to full activity at 6 weeks postoperatively, because of the decreased soft-tissue disruption. PMID:23875149

  3. Endoscopic Compartment Release for Chronic Exertional Compartment Syndrome

    PubMed Central

    Knight, Justin R.; Daniels, Marissa; Robertson, William

    2013-01-01

    Exertional compartment syndrome of the leg is a condition that can cause chronic debilitating pain in active persons during a variety of aerobic activities. Nonoperative treatments using stretching protocols and activity modifications are often unsuccessful, and thus several operative strategies have been used to treat this condition. A novel technique for endoscopically assisted fasciotomy for chronic exertional compartment syndrome is described. By use of a small laterally based incision and an arthroscope, polydioxanone sutures are passed percutaneously along the anterior and lateral compartments with the Spectrum suture-shuttling device (ConMed Linvatec, Largo, FL). These sutures are used to retract the skin and subcutaneous tissues over the respective compartments. This method allows excellent visualization of the intercompartmental septum, the superficial peroneal nerve, and all perforating vessels. The anterior and lateral compartments can be safely and completely released with this minimally invasive approach. The patient is allowed to return to full activity at 6 weeks postoperatively, because of the decreased soft-tissue disruption. PMID:23875149

  4. Dual-Compartment Inflatable Suitlock

    NASA Technical Reports Server (NTRS)

    Kennedy, Kriss J.; Guirgis, Peggy L.; Boyle, Robert M.

    2013-01-01

    There is a need for an improvement over current NASA Extravehicular Activity (EVA) technology. The technology must allow the capacity for quicker, more efficient egress/ingress, allow for shirtsleeve suit maintenance, be compact in transport, and be applicable to environments ranging from planetary surface (partial-g) to orbital or deep space zero-g environments. The technology must also be resistant to dust and other foreign contaminants that may be present on or around a planetary surface. The technology should be portable, and be capable of docking with a variety of habitats, ports, stations, vehicles, and other pressurized modules. The Dual-Compartment Inflatable Suitlock (DCIS) consists of three hard inline bulkheads, separating two cylindrical membrane-walled compartments. The Inner Bulkhead can be fitted with a variety of hatch types, docking flanges, and mating hardware, such as the Common Berthing Mechanism (CBM), for the purpose of mating with vehicles, habitats, and other pressurized modules. The Inner Bulkhead and Center Bulkhead function as the end walls of the Inner Compartment, which during operations, would stay pressurized, either matching the pressure of the habitat or acting as a lower-pressure transitional volume. The Inner Compartment contains donning/doffing fixtures and inner suit-port hatches. The Center Bulkhead has two integrated suit-ports along with a maintenance hatch. The Center Bulkhead and Outer Bulkhead function as the end walls of the Outer Compartment, which stays at vacuum during normal operations. This allows the crewmember to quickly don a suit, and egress the suitlock without waiting for the Outer Compartment to depressurize. The Outer Compartment can be pressurized infrequently for both nominal and off-nominal suit maintenance tasks, allowing shirtsleeve inspections and maintenance/repair of the environmental suits. The Outer Bulkhead has a pressure-assisted hatch door that stays open and stowed during EVA operations, but can

  5. Dual-Compartment Inflatable Suitlock

    NASA Technical Reports Server (NTRS)

    Howe, Scott; Kennedy, Kriss J.; Guirgis, Peggy L.

    2012-01-01

    A paper discusses a dual-compartment inflatable suitlock (DCIS) for Extra - vehicular Activity (EVA) that will allow for dust control, suit maintenance, and efficient EVA egress/ingress. The expandable (inflatable technologies) aspect of the design will allow the unit to stow in a compact package for transport. The DCIS consists of three hard, in line bulkheads, separating two cylindrical membrane-walled compartments. The inner bulkhead can be fitted with a variety of hatch types, docking flanges, and mating hardware, such as the common berthing mechanism (CBM), for the purpose of mating with vehicles, habitats, and other pressurized modules. The inner bulkhead and center bulkhead function as the end walls of the inner compartment, which, during operations, would stay pressurized, either matching the pressure of the habitat or acting as a lower-pressure transitional volume. The suited crewmember can quickly don a suit, and egress the suitlock without waiting for the compartment to depressurize. The outer compartment can be pressurized infrequently, when a long dwell time is expected prior to the next EVA, or during off-nominal suit maintenance tasks, allowing shirtsleeve inspections and maintenance of the space suits. The outer bulkhead has a pressure-assisted hatch door that stays open and stowed routinely, but can be closed for suit maintenance and pressurization as needed.

  6. Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA.

    PubMed

    Karlsson, Oskar; Berg, Anna-Lena; Hanrieder, Jörg; Arnerup, Gunnel; Lindström, Anna-Karin; Brittebo, Eva B

    2015-03-01

    The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo. PMID:24798087

  7. Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.

    PubMed

    Vernet, Erik; Lundberg, Emma; Friedman, Mikaela; Rigamonti, Nicolò; Klausing, Sandra; Nygren, Per-Ake; Gräslund, Torbjörn

    2009-09-01

    Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate. PMID:19552886

  8. Discrepancy in calcium release from the sarcoplasmic reticulum and intracellular acidic stores for the protection of the heart against ischemia/reperfusion injury.

    PubMed

    Khalaf, Aseel; Babiker, Fawzi

    2016-09-01

    We and others have demonstrated a protective effect of pacing postconditioning (PPC) against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection are not completely clear. In the present study, we evaluated the effects of calcium release from the sarcoplasmic reticulum (SR) and the novel intracellular acidic stores (AS). Isolated rat hearts (n = 6 per group) were subjected to coronary occlusion followed by reperfusion using a modified Langendorff system. Cardiac hemodynamics and contractility were assessed using a data acquisition program, and cardiac injury was evaluated by creatine kinase (CK) and lactate dehydrogenase (LDH) levels. Hearts were subjected to 30 min of regional ischemia, produced by ligation of the left anterior descending (LAD) coronary artery, followed by 30 min of reperfusion. The hearts were also subjected to PPC (3 cycles of 30 s of left ventricle (LV) pacing alternated with 30 s of right atrium (RA) pacing) and/or were treated during reperfusion with agonists or antagonists of release of calcium from SR or AS. PPC significantly (P < 0.05) normalized LV, contractility, and coronary vascular dynamics and significantly (P < 0.001) decreased heart enzyme levels compared to the control treatments. The blockade of SR calcium release resulted in a significant (P < 0.01) recovery in LV function and contractility and a significant reduction in CK and LDH levels (P < 0.01) when applied alone or in combination with PPC. Interestingly, the release of calcium from AS alone or in combination with PPC significantly improved LV function and contractility (P < 0.05) and significantly decreased the CK and LDH levels (P < 0.01) compared to the control treatments. An additive effect was produced when agonism of calcium release from AS or blockade of calcium release from the SR was combined with PPC. Calcium release from AS and blockade of calcium release from the SR protect the heart against I

  9. 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

  10. Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism.

    PubMed

    Georgiou, Dimitra K; Dagnino-Acosta, Adan; Lee, Chang Seok; Griffin, Deric M; Wang, Hui; Lagor, William R; Pautler, Robia G; Dirksen, Robert T; Hamilton, Susan L

    2015-09-25

    Ca(2+) permeation and/or binding to the skeletal muscle L-type Ca(2+) channel (CaV1.1) facilitates activation of Ca(2+)/calmodulin kinase type II (CaMKII) and Ca(2+) store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca(2+) binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle. PMID:26245899

  11. Intracellular trafficking of silicon particles and logic-embedded vectors

    NASA Astrophysics Data System (ADS)

    Ferrati, Silvia; Mack, Aaron; Chiappini, Ciro; Liu, Xuewu; Bean, Andrew J.; Ferrari, Mauro; Serda, Rita E.

    2010-08-01

    Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 μm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon

  12. 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

  13. Simultaneous pH measurement in endocytic and cytosolic compartments in living cells using confocal microscopy.

    PubMed

    Lucien, Fabrice; Harper, Kelly; Pelletier, Pierre-Paul; Volkov, Leonid; Dubois, Claire M

    2014-01-01

    Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can

  14. The Orbital Workshop Shower Compartment

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This photograph shows technicians performing a checkout of the Metabolic Analyzer (center background) and the Ergometer (foreground) in the Orbital Workshop (OWS). The shower compartment is at right. The Ergometer (Skylab Experiment M171) evaluated man's metabolic effectiveness and cost of work in space environment. Located in the experiment and work area of the OWS, the shower compartment was a cylindrical cloth enclosure that was folded flat when not in use. The bottom ring of the shower was fastened to the floor and contained foot restraints. The upper ring contained the shower head and hose. To use the shower, the astronaut filled a pressurized portable bottle with heated water and attached the bottle to the ceiling. A flexible hose cornected the water bottle to a handheld shower head. The astronaut pulled the cylindrical shower wall up into position and bathed, using liquid soap. Both soap and water were carefully rationed, having been premeasured for economical use.

  15. The Orbital Workshop Shower Compartment

    NASA Technical Reports Server (NTRS)

    1972-01-01

    In this photograph, the Orbital Workshop shower compartment was unfolded by technicians for inspection. The shower compartment was a cylindrical cloth enclosure that was folded flat when not in use. The bottom ring of the shower was fastened to the floor and contained foot restraints. The upper ring contained the shower head and hose. To use the shower, the astronaut filled a pressurized portable bottle with heated water and attached the bottle to the ceiling. A flexible hose cornected the water bottle to a handheld shower head. The astronaut pulled the cylindrical shower wall up into position and bathed, using liquid soap. Both soap and water were carefully rationed, having been premeasured for economical use.

  16. The Orbital Workshop Sleep Compartment

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This wide-angle view is of the Orbital Workshop (OWS) sleep compartment, located in the lower level of the OWS. Each crewman was assigned a small space for sleeping and zipped themselves into sleeping bags stretched against the wall. Because of the absence of gravity, sleeping comfort was achieved in any position relative to the spacecraft; body support was not necessary. Sleeping could be accommodated quite comfortably in a bag that held the body at a given place in Skylab.

  17. Method and apparatus to assess compartment syndrome

    NASA Technical Reports Server (NTRS)

    Ueno, Toshiaki (Inventor); Hargens, Alan R. (Inventor); Yost, William T. (Inventor)

    2008-01-01

    A method and apparatus for measuring pressure buildup in a body compartment that encases muscular tissue. The method includes assessing the body compartment configuration and identifying the effect of pulsatile components on at least one compartment dimension. This process is used in preventing tissue necrosis, and in decisions of whether to perform surgery on the body compartment for prevention of Compartment Syndrome. An apparatus is used for measuring excess pressure in the body compartment having components for imparting ultrasonic waves such as a transducer, placing the transducer to impart the ultrasonic waves, capturing the reflected imparted ultrasonic waves, and converting them to electrical signals, a pulsed phase-locked loop device for assessing a body compartment configuration and producing an output signal, and means for mathematically manipulating the output signal to thereby categorize pressure build-up in the body compartment from the mathematical manipulations.

  18. Control system maintains compartment at constant temperature

    NASA Technical Reports Server (NTRS)

    Lindberg, J. G.

    1966-01-01

    Gas-filled permeable insulating material maintains an enclosed compartment at a uniform temperature. The material is interposed between the two walls of a double-walled enclosure surrounding the compartment.

  19. 14 CFR 29.853 - Compartment interiors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Compartment interiors. 29.853 Section 29.853 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Design and Construction Fire Protection § 29.853 Compartment interiors. For each compartment to...

  20. Forearm Compartment Syndrome Caused by Reperfusion Injury

    PubMed Central

    Sayar, Ufuk; Mataracı, İlker

    2014-01-01

    Compartment syndrome is commonly seen following lower extremity ischemia. However, upper extremities' compartment syndrome, especially after any vascular surgical procedures, is infrequent. Herein we report a case of an acute forearm compartment syndrome that was developed after delayed brachial artery embolectomy. PMID:25120938

  1. 49 CFR 38.127 - Sleeping compartments.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Sleeping compartments. 38.127 Section 38.127... SPECIFICATIONS FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems § 38.127 Sleeping compartments. (a) Sleeping compartments required to be accessible shall be designed so as to allow a person using...

  2. 49 CFR 38.127 - Sleeping compartments.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Sleeping compartments. 38.127 Section 38.127... SPECIFICATIONS FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems § 38.127 Sleeping compartments. (a) Sleeping compartments required to be accessible shall be designed so as to allow a person using...

  3. 49 CFR 38.127 - Sleeping compartments.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Sleeping compartments. 38.127 Section 38.127... SPECIFICATIONS FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems § 38.127 Sleeping compartments. (a) Sleeping compartments required to be accessible shall be designed so as to allow a person using...

  4. 49 CFR 38.127 - Sleeping compartments.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Sleeping compartments. 38.127 Section 38.127... SPECIFICATIONS FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems § 38.127 Sleeping compartments. (a) Sleeping compartments required to be accessible shall be designed so as to allow a person using...

  5. 14 CFR 23.771 - Pilot compartment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment. 23.771 Section 23.771... Cargo Accommodations § 23.771 Pilot compartment. For each pilot compartment— (a) The compartment and its equipment must allow each pilot to perform his duties without unreasonable concentration or fatigue;...

  6. 14 CFR 27.771 - Pilot compartment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment. 27.771 Section 27.771... Pilot compartment. For each pilot compartment— (a) The compartment and its equipment must allow each pilot to perform his duties without unreasonable concentration or fatigue; (b) If there is provision...

  7. 14 CFR 25.771 - Pilot compartment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment. 25.771 Section 25.771... Pilot compartment. (a) Each pilot compartment and its equipment must allow the minimum flight crew... pilot, the airplane must be controllable with equal safety from either pilot seat. (d) The...

  8. 14 CFR 29.771 - Pilot compartment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment. 29.771 Section 29.771... Pilot compartment. For each pilot compartment— (a) The compartment and its equipment must allow each pilot to perform his duties without unreasonable concentration or fatigue; (b) If there is provision...

  9. 14 CFR 29.771 - Pilot compartment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment. 29.771 Section 29.771... Pilot compartment. For each pilot compartment— (a) The compartment and its equipment must allow each pilot to perform his duties without unreasonable concentration or fatigue; (b) If there is provision...

  10. 14 CFR 27.771 - Pilot compartment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment. 27.771 Section 27.771... Pilot compartment. For each pilot compartment— (a) The compartment and its equipment must allow each pilot to perform his duties without unreasonable concentration or fatigue; (b) If there is provision...

  11. 49 CFR 38.127 - Sleeping compartments.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Sleeping compartments. 38.127 Section 38.127... SPECIFICATIONS FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems § 38.127 Sleeping compartments. (a) Sleeping compartments required to be accessible shall be designed so as to allow a person using...

  12. Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.

    PubMed

    Yang, Maiyun; Jalloh, Abubakar S; Wei, Wei; Zhao, Jing; Wu, Peng; Chen, Peng R

    2014-01-01

    Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions. PMID:25236616

  13. Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin.

    PubMed

    Yoshida, Aya; Matumoto, Makoto; Hshizume, Hiroyuki; Oba, Yoshiro; Tomishige, Tatuo; Inagawa, Hiroyuki; Kohchi, Chie; Hino, Mami; Ito, Fuminori; Tomoda, Keishiro; Nakajima, Takehisa; Makino, Kimiko; Terada, Hiroshi; Hori, Hitoshi; Soma, Gen-Ichiro

    2006-08-01

    Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages. PMID:16879999

  14. Chronic Exertional Compartment Syndrome Testing.

    PubMed

    Flick, David; Flick, Renee

    2015-01-01

    Chronic exertional compartment syndrome is diagnosed based on historical and physical exam findings combined with elevated intracompartmental pressures. Direct static testing with a large bore needle device is the most common instrument used for diagnosis. Based on the most recent systematic reviews, there is poor evidence for the traditional diagnostic pressures used in practice with no standardization of the procedure. New research has introduced a standardized approach with dynamic testing of the limb with transducer-tipped catheters. Less invasive methods of testing using radiologic techniques are currently under investigation. A detailed understanding of the anatomy and physiology of the limb is paramount in executing a safe and accurate procedure. PMID:26359839

  15. A general mechanism for intracellular toxicity of metal-containing nanoparticles.

    PubMed

    Sabella, Stefania; Carney, Randy P; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M; Bakr, Osman M; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-06-21

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment--where particles are abundantly internalized--is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. PMID:24842463

  16. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

    PubMed Central

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  17. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

    PubMed

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  18. 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

  19. pH-responsive PDMS-b-PDMAEMA micelles for intracellular anticancer drug delivery.

    PubMed

    Car, Anja; Baumann, Patric; Duskey, Jason T; Chami, Mohamed; Bruns, Nico; Meier, Wolfgang

    2014-09-01

    A series of poly(dimethysiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer radical polymerization (ATRP). In aqueous solution the polymers self-assembled into micelles with diameters between 80 and 300 nm, with the ability to encapsulate DOX. The polymer with the shortest PDMAEMA block (5 units) displayed excellent cell viability, while micelles containing longer PDMAEMA block lengths (13 and 22 units) led to increased cytotoxicity. The carriers released DOX in response to a decrease in pH from 7.4 to 5.5. Confocal laser scanning microscopy (CLSM) revealed that nanoparticles were taken up by endocytosis into acidic cell compartments. Furthermore, DOX-loaded nanocarriers exhibited intracellular pH-response as changes in cell morphology and drug release were observed within 24 h. PMID:25068477

  20. A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments.

    PubMed

    Zhang, M; Søndergaard, R V; Kumar, E K P; Henriksen, J R; Cui, D; Hammershøj, P; Clausen, M H; Andresen, T L

    2015-11-01

    Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements of acidic intracellular pH (pH < 4) in living cells are still challenging due to the lack of suitable nanosensors. In this paper we successfully developed a multiple sensor, a fluorophore based nanosensor, with an unprecedented broad measurement range from pH 1.4 to 7.0. In this nanosensor, three pH-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured without being too close to the limits of its pH-range. The nanosensor exhibits no susceptibility to interference by other intracellular ions at physiological concentrations. Due to its positive surface charge it is spontaneously internalized by HeLa cells and localizes to the lysosomes where the mean pH was measured at 4.6. This quadruple-labelled nanosensor performs accurate measurements of fluctuations of lysosomal pH in both directions, which was shown by treatment with the V-ATPase inhibitor bafilomycin A1 or its substrate ATP in HeLa cells. These measurements indicate that this novel quadruple-labelled nanosensor is a promising new tool for measuring the pH of acidic compartments in living cells. PMID:26393332

  1. 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

  2. Independent Active Contraction of Extraocular Muscle Compartments

    PubMed Central

    Shin, Andrew; Yoo, Lawrence; Demer, Joseph L.

    2015-01-01

    Purpose. Intramuscular innervation of horizontal rectus extraocular muscle (EOMs) is segregated into superior and inferior (transverse) compartments, whereas all EOMs are also divided into global (GL) and orbital (OL) layers with scleral and pulley insertions, respectively. Mechanical independence between both types of compartments has been demonstrated during passive tensile loading. We examined coupling between EOM compartments during active, ex vivo contraction. Methods. Fresh bovine EOMs were removed, and one compartment of each was coated with hydrophobic petrolatum. Contraction of the uncoated compartment was induced by immersion in a solution of 50 mM CaCl2 at 38°C labeled with sodium fluorescein dye, whereas tensions in both compartments were monitored by strain gauges. Control experiments omitted petrolatum so that the entire EOM contracted. After physiological experiments, EOMs were sectioned transversely to demonstrate specificity of CaCl2 permeation by yellow fluorescence dye excited by blue light. Results. In control experiments without petrolatum, both transverse and GL and OL compartments contracted similarly. Selective compartmental omission of petrolatum caused markedly independent compartmental contraction whether measured at the GL or the OL insertions or for transverse compartments at the scleral insertion. Although some CaCl2 spread occurred, mean (±SD) tension in the coated compartments averaged only 10.5 ± 3.3% and 6.0 ± 1.5% in GL/OL and transverse compartments, respectively relative to uncoated compartments. Fluorescein penetration confirmed selective CaCl2 permeation. Conclusions. These data confirm passive tensile findings of mechanical independence of EOM compartments and extend results to active contraction. EOMs behave actively as if composed of mechanically independent parallel fiber bundles having different insertional targets, consistent with the active pulley and transverse compartmental hypotheses. PMID:25503460

  3. Decorporation of Pu/Am Actinides by Chelation Therapy: New Arguments in Favor of an Intracellular Component of DTPA Action.

    PubMed

    Grémy, Olivier; Laurent, David; Coudert, Sylvie; Griffiths, Nina M; Miccoli, Laurent

    2016-06-01

    Diethylenetriaminepentaacetic acid (DTPA) is currently still the only known chelating drug that can be used for decorporation of internalized plutonium (Pu) and americium (Am). It is generally assumed that chelation occurs only in biological fluids, thus preventing Pu/Am deposition in target tissues. We postulate that actinide chelation may also occur inside cells by a mechanism called "intracellular chelation". To test this hypothesis, rats were given DTPA either prior to (termed "prophylactic" treatment) or belatedly after (termed "delayed" treatment) Pu/Am injection. DTPA decorporation efficacy was systematically tested for both plutonium and americium. Both prophylactic and delayed DTPA elicited marked decreases in liver Pu/Am. These results can be explained by chelation within subcellular compartments where DTPA efficacy increased as a function of a favorable intracellular DTPA-to-actinide molar ratio. The efficacy of intracellular chelation of liver actinides decreased with the delay of treatment. This is probably explained by progressive actinide binding to the high-affinity ligand ferritin followed by migration to lysosomes. Intracellular chelation was reduced as the gap between prophylactic treatment and contamination increased. This may be explained by the reduction of the intracellular DTPA pool, which declined exponentially with time. Skeletal Pu/Am was also reduced by prophylactic and delayed DTPA treatments. This decorporation of bone actinides may mainly result from extracellular chelation on bone surfaces. This work provides converging evidence for the involvement of an intracellular component of DTPA action in the decorporation process. These results may help to improve the interpretation of biological data from DTPA-treated contamination cases and could be useful to model DTPA therapy regimens. PMID:27195610

  4. 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

  5. Modeling the effects of sodium chloride, acetic acid and intracellular pH on the survival of Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primar...

  6. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

    PubMed Central

    Alconada, A; Bauer, U; Hoflack, B

    1996-01-01

    We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms. Images PMID:8947032

  7. Protective effects of Alpha-lipoic acid on MeHg-induced oxidative damage and intracellular Ca(2+) dyshomeostasis in primary cultured neurons.

    PubMed

    Yang, Tianyao; Xu, Zhaofa; Liu, Wei; Xu, Bin; Deng, Yu

    2016-05-01

    Methylmercury (MeHg) is one of the ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. However, the mechanisms of MeHg-induced neuronal cell death are incompletely understood. Treatment of neuronal cells with MeHg (0-2 μM) for 0.5-12 h, or pretreated with LA (12.5-100 μM) for 0.5-6 h resulted in toxic effects of primary cultured neurons concentration- and time-dependently. For further experiments, 12.5, 25, and 50 μM of LA pretreatment for 3 h followed by 1 μM MeHg for 6 h were performed for the examination of the responses of neurons. Exposure of MeHg resulted in damages of neurons, which were shown by a loss of cell viability, and supported by high levels of lactate dehydrogenase (LDH) release, apoptosis, and morphological changes. In addition, neurons were sensitive to MeHg-mediated oxidative stress, a finding that is consistent with ROS over-production, leading to decrease Ca(2+)-ATPase activity and increase intracellular free calcium. Moreover, expressions of NMDA receptor subunits in neurons were down-regulated after MeHg exposure, and expression of NR2A mRNA and protein were much more sensitive to MeHg than those of NR1 and NR2B. On the contrary, pretreatment with LA presented a concentration-dependent prevention against MeHg-mediated cytotoxic effects of neurons. In conclusion, present results showed that oxidative stress and intracellular Ca(2+ )dyshomeostasis resulting from MeHg exposure contributed to neuronal injury. LA could attenuate MeHg-induced neuronal toxicity via its antioxidant properties in primary cultured neurons. PMID:26986620

  8. 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.

  9. Chronic exertional compartment syndrome of the superficial posterior compartment: Soleus syndrome

    PubMed Central

    Gross, Christopher E; Parekh, Bela J; Adams, Samuel B; Parekh, Selene G

    2015-01-01

    Chronic exertional compartment syndrome (CECS) represents the second most-common cause of exertional leg pain with incidence of 27-33%. CECS of the superficial posterior compartment, or soleus syndrome, is rare and has only been discussed briefly in the literature. We discuss the management of two patients with bilateral soleus syndrome or CECS of the superficial posterior compartment. PMID:26538766

  10. Chronic exertional compartment syndrome of the superficial posterior compartment: Soleus syndrome.

    PubMed

    Gross, Christopher E; Parekh, Bela J; Adams, Samuel B; Parekh, Selene G

    2015-01-01

    Chronic exertional compartment syndrome (CECS) represents the second most-common cause of exertional leg pain with incidence of 27-33%. CECS of the superficial posterior compartment, or soleus syndrome, is rare and has only been discussed briefly in the literature. We discuss the management of two patients with bilateral soleus syndrome or CECS of the superficial posterior compartment. PMID:26538766

  11. 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

  12. 14 CFR 25.787 - Stowage compartments.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Stowage compartments. 25.787 Section 25.787 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS... equipment (such as life rafts), and any other stowage compartment must be designed for its placarded...

  13. 14 CFR 25.787 - Stowage compartments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Stowage compartments. 25.787 Section 25.787 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS... equipment (such as life rafts), and any other stowage compartment must be designed for its placarded...

  14. 14 CFR 25.787 - Stowage compartments.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Stowage compartments. 25.787 Section 25.787 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS... equipment (such as life rafts), and any other stowage compartment must be designed for its placarded...

  15. 14 CFR 25.787 - Stowage compartments.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Stowage compartments. 25.787 Section 25.787 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS... equipment (such as life rafts), and any other stowage compartment must be designed for its placarded...

  16. 14 CFR 25.787 - Stowage compartments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stowage compartments. 25.787 Section 25.787 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Design and Construction Personnel and Cargo Accommodations § 25.787 Stowage compartments. (a)...

  17. Membrane contact sites between pathogen-containing compartments and host organelles.

    PubMed

    Dumoux, Maud; Hayward, Richard D

    2016-08-01

    Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid transport between pathogen-containing compartments and host organelles, including the formation of static membrane contact sites. We discuss how lipid scavenging can be mediated via the reprogramming of cellular transporters at these interfaces and describe recent data suggesting that pathogen effectors modulate the formation of specific membrane contacts. Further study of these emerging mechanisms is likely to yield new insights into the cell biology of lipid transport and organelle communication, which highlights potential new targets and strategies for future therapeutics. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26825687

  18. How paramagnetic and diamagnetic LMOCs detect picric acid from surface water and the intracellular environment: a combined experimental and DFT-D3 study.

    PubMed

    Ghosh, Pritam; Banerjee, Priyabrata

    2016-08-17

    Diamagnetic and Paramagnetic Luminescent Metal Organic Complexes (LMOCs) have been reported for Explosive and Pollutant Nitro Aromatic (epNAC) recognition. The diamagnetic complex shows a highly intense AIE induced by NEt3H(+), which disappears after picric acid recognition and subsequently RET will quench the emission intensity. Radical stabilized paramagnetic LMOCs seem to be active but show lower sensing efficiency in comparison with diamagnetic LMOCs. Solution and solid state spectroscopy studies along with DFT-D3 have been executed to enlighten the host guest interaction. Limit of PA detection is ∼250 ppb with a binding constant of 1.2 × 10(5) M(-1). Time-stepping, i.e. intervening in the problem of picric acid recognition from surface water collected from several places of West Bengal, India, has been performed. Mutagenic picric acid has been successfully detected in an aqueous medium inside both prokaryotic and eukaryotic cells at a ppm level using fluorescence microscopy. PMID:27171212

  19. Malate-aspartate shuttle inhibitor aminooxyacetic acid leads to decreased intracellular ATP levels and altered cell cycle of C6 glioma cells by inhibiting glycolysis.

    PubMed

    Wang, Caixia; Chen, Heyu; Zhang, Mingchao; Zhang, Jie; Wei, Xunbin; Ying, Weihai

    2016-08-01

    NADH shuttles, including malate-aspartate shuttle (MAS) and glycerol-3-phosphate shuttle, can shuttle the reducing equivalents of cytosolic NADH into mitochondria. It is widely accepted that the major function of NADH shuttles is to increase mitochondrial energy production. Our study tested the hypothesis that the novel major function of NADH shuttles in cancer cells is to maintain glycolysis by decreasing cytosolic NADH/NAD(+) ratios. We found that AOAA, a widely used MAS inhibitor, led to decreased intracellular ATP levels, altered cell cycle and increased apoptosis and necrosis of C6 glioma cells, without affecting the survival of primary astrocyte cultures. AOAA also decreased the glycolytic rate and the levels of extracellular lactate and pyruvate, without affecting the mitochondrial membrane potential of C6 cells. Moreover, the toxic effects of AOAA were completely prevented by pyruvate treatment. Collectively, our study has suggested that AOAA may be used to selectively decrease glioma cell survival, and the major function of MAS in cancer cells may be profoundly different from its major function in normal cells: The major function of MAS in cancer cells is to maintain glycolysis, instead of increasing mitochondrial energy metabolism. PMID:27157912

  20. 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

  1. Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides.

    PubMed

    Weng, Alexander; Manunta, Maria D I; Thakur, Mayank; Gilabert-Oriol, Roger; Tagalakis, Aristides D; Eddaoudi, Ayad; Munye, Mustafa M; Vink, Conrad A; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F; Hart, Stephen L

    2015-05-28

    Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems. PMID:25758332

  2. Multifunctional Mesoporous Silica Nanoparticles Based on Charge-Reversal Plug-Gate Nanovalves and Acid-Decomposable ZnO Quantum Dots for Intracellular Drug Delivery.

    PubMed

    Zhang, Jing; Wu, Dan; Li, Meng-Fei; Feng, Jie

    2015-12-01

    A novel type of pH-responsive multifunctional mesoporous silica nanoparticle (MSN) was developed for cancerous cells drug delivery and synergistic therapy of tumor. MSNs were covered with a kind of cell-penetrating peptide, deca-lysine sequence (K10), to enhance their escape from the endosomes. After K10's primary amines were reacted with citraconic anhydride to form acid-labile β-carboxylic amides, zinc oxide (ZnO) quantum dots (QDs) were introduced to cap MSNs via electrostatic interaction. The obtained ZnO@MSN drug-delivery system (DDS) achieves "zero-premature" drug release under a physiological environment. However, once the DDS is transferred to the cancerous cells' acidic endosome, ZnO QDs would rapidly dissolve and the acid-labile amides on the side chain of K10 would hydrolyze to regenerate primary amines, resulting in the uncapping of MSNs and exposure of the cell-penetrating peptide K10. The regenerated K10 could help the DDS escape from the endosome and efficiently release the loaded drugs inside the cells. At the meantime, because of the cytotoxicity of ZnO QDs at their destination, the ZnO@MSN DDS may achieve a synergistic antitumor effect to improve the therapeutic index. PMID:26553405

  3. 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

  4. Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

    PubMed Central

    Craig, R K; Boulton, A P; Harrison, O S; Parker, D; Campbell, P N

    1979-01-01

    (pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland. Images PLATE 1 Fig. 2. Fig. 3. Fig. 10. Fig. 11. PMID:518553

  5. 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

  6. 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

  7. Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

    PubMed Central

    Liu, Yanhua; Zhang, Qiufeng; Hu, Mo; Yu, Kaiwen; Fu, Jiaqi; Zhou, Fan

    2015-01-01

    Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways. PMID:25939512

  8. 14 CFR 29.853 - Compartment interiors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... and acoustical insulation and insulation covering, air ducting, joint and edge covering, cargo compartment liners, insulation blankets, cargo covers, and transparencies, molded and thermoformed parts, air... in a common housing, seat belts, shoulder harnesses, and cargo and baggage tiedown...

  9. Microscale and nanoscale compartments for biotechnology.

    PubMed

    Retterer, Scott T; Simpson, Michael L

    2012-08-01

    Compartmentalization is essential in the organization of biological systems, playing a fundamental role in modulating biochemical activity. An appreciation of the impact that biological compartments have on chemical reactions and an understanding of the physical and chemical phenomena that affect their assembly and function have inspired the development of synthetic compartments. Organic compartments assembled from amphiphilic molecules or derived from biological materials, have formed the basis of initial work in the field. However, inorganic and hybrid organic-inorganic compartments that capitalize on the optical and catalytic properties of metal and semiconductor materials are emerging. Methods for arraying these microcompartment and nanocompartment materials in higher order systems promise to enable the scaling and integration of these technologies for industrial and commercial applications. PMID:22321942

  10. Compartment syndrome after tibial plateau fracture☆

    PubMed Central

    Pitta, Guilherme Benjamin Brandão; dos Santos, Thays Fernanda Avelino; dos Santos, Fernanda Thaysa Avelino; da Costa Filho, Edelson Moreira

    2014-01-01

    Fractures of the tibial plateau are relatively rare, representing around 1.2% of all fractures. The tibia, due to its subcutaneous location and poor muscle coverage, is exposed and suffers large numbers of traumas, not only fractures, but also crush injuries and severe bruising, among others, which at any given moment, could lead compartment syndrome in the patient. The case is reported of a 58-year-old patient who, following a tibial plateau fracture, presented compartment syndrome of the leg and was submitted to decompressive fasciotomy of the four right compartments. After osteosynthesis with internal fixation of the tibial plateau using an L-plate, the patient again developed compartment syndrome. PMID:26229779

  11. Aircraft Cargo Compartment Fire Test Simulation Program

    NASA Technical Reports Server (NTRS)

    Blumke, R. E.

    1977-01-01

    The objective of the test was to assess fire containment and fire extinguishment in the cargo by reducing the ventilation through the cargo compartment. Parameters which were measured included ignition time, burnthrough time, and physical damage to the cargo liner, composition of selected combustible gases, temperature-time histories, heat flux, and detector response. The ignitor load was made of a typical cargo consisting of filled cardboard cartons occupying 50% of the compartment volume.

  12. Free Fatty Acids Increase Intracellular Lipid Accumulation and Oxidative Stress by Modulating PPARα and SREBP-1c in L-02 Cells.

    PubMed

    Qin, Shumin; Yin, Jinjin; Huang, Keer

    2016-07-01

    Excessive fat accumulation and increased oxidative stress contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, the mechanisms underlying the development of steatosis are not entirely understood. The present study was undertaken to establish an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the effects of oxidative stress could be studied in L-02 cells. We investigated the effects of free fatty acids (FFA) (palmitate:oleate, 1:2) on lipid accumulation and oxidative stress and their possible mechanisms in L-02 cells. High concentrations of fatty acids significantly induced excessive lipid accumulation and oxidative stress in L-02 cells, which could only be reversed with 50 μΜ WY14643 (the PPARα agonist). Immunoblotting and qPCR analyses revealed that FFA downregulated the expression of proliferator-activated receptor alpha (PPARα), which contributed to the increased activation of sterol regulatory element binding protein-1c (SREBP-1c). These results suggest that FFA induce lipid accumulation and oxidative stress in L-02 cells by upregulating SREBP-1c expression through the suppression of PPARα. PMID:27270405

  13. INTRACELLULAR ANTIOXIDANT ACTIVITY OF A STREPTOMYCES SP. 8812 SECONDARY METABOLITE, 6,7-DIHYDROXY-3,4-DIHYDROISOQINO- LINE-3-CARBOXYLIC ACID, AND ITS SYNTHETIC DERIVATIVES.

    PubMed

    Guśpiel, Adam; Ziemska, Joanna; Cześcik, Agnieszka; Kawecki, Robert; Solecka, Jolanta

    2016-01-01

    The aim of this study was to determine the antioxidant properties of 6,7-dihydroxy-3,4-dihydroiso- quinoline-3-carboxylic acid (1) and its derivatives in living cells against reactive forms of oxygen and nitrogen, i.e., hydrogen peroxide and nitric oxide. Four of tested compounds scavenged the reactive form of nitrogen more efficiently or similarly to Trolox (EC50 = 55.80 µM). Two compounds exhibited antioxidant activity against reactive oxygen species better than Trolox (EC50 = 51.88 µM). The most active derivative of 1 was the compound containing an iodine atom at position 8 (6,7-dihydroxy-8-iodo-3,4-dihydroisoquinoline-3-carboxylic acid). Our studies showed that some of the derivatives had the ability to cross the cell membrane and scavenge free radicals inside living cells. Thus, they are able to protect DNA and other cellular structures from the dam- aging effects of reactive oxygen and nitrogen species. In addition, some molecular descriptors of the tested compounds were determined with the use of ICM Pro (Molsoft L.L.C.). PMID:27476282

  14. A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.

    PubMed Central

    Partanen, Sanna; Storch, Stephan; Löffler, Hans-Gerhard; Hasilik, Andrej; Tyynelä, Jaana; Braulke, Thomas

    2003-01-01

    The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme. PMID:12350228

  15. 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

  16. Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

    PubMed Central

    Hansen, M; Jelinek, L; Jones, R S; Stegeman-Olsen, J; Barklis, E

    1993-01-01

    Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections. Images PMID:8350394

  17. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    NASA Astrophysics Data System (ADS)

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-05-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where particles are abundantly internalized - is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a ``lysosome-enhanced Trojan horse effect'' since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where

  18. Redox-sensitive YFP sensors for monitoring dynamic compartment-specific glutathione redox state.

    PubMed

    Banach-Latapy, Agata; He, Tiantian; Dardalhon, Michèle; Vernis, Laurence; Chanet, Roland; Huang, Meng-Er

    2013-12-01

    Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. PMID:23891676

  19. 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

  20. Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments.

    PubMed

    Waseem, Tatyana; Mukhtarov, Marat; Buldakova, Svetlana; Medina, Igor; Bregestovski, Piotr

    2010-10-30

    Chloride (Cl) participates in a variety of physiological functions. To study processes connected with Cl homeostasis we need effective and quantitative probes allowing measurements of intracellular Cl concentration ([Cl(-)](i)) in different cell types, particularly in specialized small cellular compartments such as dendrites and dendritic spines. Of the different tools proposed for monitoring [Cl(-)](i), the genetically encoded Cl-sensitive indicators are the most promising. Recently, a ratiometric CFP-YFP based construct, termed "Cl-Sensor", with a relatively high sensitivity to Cl has been proposed (Markova et al., 2008). In the present study, we have developed conditions for the efficient expression of Cl-Sensor in tiny neuronal compartments including distal dendrites and spines. We also propose a new approach for the calibration of intracellularly expressed probes using a natural triterpenoid saponin, β-escin. We have mapped [Cl(-)](i) distribution in different neuronal compartments of cultured hippocampal and spinal cord neurons. The maximum Cl concentration was observed in the soma and it had a tendency to decrease gradually along dendritic branches, reaching minimum values in thin distal dendrites. We have also monitored transient increases in intracellular Cl in dendritic spines caused by glutamate application. These results demonstrate that Cl-Sensor enables non-invasive monitoring of the [Cl(-)](i) distribution in different types of neurons with variable morphology. This probe represents an effective tool for the quantitative estimation of [Cl(-)](i) in various cellular compartments including dendritic spines. PMID:20705097

  1. Delayed Presentation of Acute Gluteal Compartment Syndrome

    PubMed Central

    Tasch, James J.; Misodi, Emmanuel O.

    2016-01-01

    Patient: Male, 23 Final Diagnosis: Acute gluteal compartment syndrome Symptoms: — Medication: — Clinical Procedure: Gluteal fasciotomy Specialty: Critical Care Medicine Objective: Unusual clinical course Background: Acute gluteal compartment syndrome is a rare condition that usually results from prolonged immobilization following a traumatic event, conventionally involving the presence of compounding factors such as alcohol or opioid intoxication. If delay in medical treatment is prolonged, severe rhabdomyolysis may ensue, leading to acute renal failure and potentially death. Case Report: We report the case of a 23-year-old male with a recent history of incarceration and recreational drug use, who presented with reports of severe right-sided buttock pain and profound right-sided neurological loss following a questionable history involving prolonged immobilization after a fall from a standing position. The patient required an emergent gluteal fasciotomy immediately upon admission and required temporary hemodialysis. After an extended hospital stay, he ultimately recovered with only mild deficits in muscular strength in the right lower extremity. Conclusions: This report demonstrates the importance of early recognition of gluteal compartment syndrome to prevent morbidity and mortality. Compartment syndrome presents in many unique ways, and healthcare practitioners must have a keen diagnostic sense to allow for early surgical intervention. Proper wick catheter measurements should be utilized more frequently, instead of relying on clinical symptomatology such as loss of peripheral pulses for diagnosis of compartment syndrome. PMID:27432320

  2. 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

  3. Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment.

    PubMed

    Malhotra, Himanshu; Sheokand, Navdeep; Kumar, Santosh; Chauhan, Anoop S; Kumar, Manoj; Jakhar, Priyanka; Boradia, Vishant M; Raje, Chaaya I; Raje, Manoj

    2016-05-01

    Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle. PMID:27305829

  4. Post-dialysis urea concentration: comparison between one- compartment model and two-compartment model

    NASA Astrophysics Data System (ADS)

    Tamrin, N. S. Ahmad; Ibrahim, N.

    2014-11-01

    The reduction of the urea concentration in blood can be numerically projected by using one-compartment model and two-compartment model with no variation in body fluid. This study aims to compare the simulated values of post-dialysis urea concentration for both models with the clinical data obtained from the hospital. The clinical assessment of adequacy of a treatment is based on the value of Kt/V. Further, direct calculation using clinical data and one-compartment model are presented in the form of ratio. It is found that the ratios of postdialysis urea concentration simulated using two-compartment model are higher compared to the ratios of post-dialysis urea concentration using one-compartment model. In addition, most values of post-dialysis urea concentration simulated using two-compartment model are much closer to the clinical data compared to values simulated using one-compartment model. Kt/V values calculated directly using clinical data are found to be higher than Kt/V values derived from one-compartment model.

  5. Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two- and a novel three-compartment scale-down bioreactor.

    PubMed

    Lemoine, Anja; Maya Martίnez-Iturralde, Nina; Spann, Robert; Neubauer, Peter; Junne, Stefan

    2015-06-01

    The oscillatory conditions in substrate and oxygen supply that typically occur on a large (industrial) scale are usually simulated in two-compartment scale-down reactors. In this study, the performance of nutrient-limited fed-batch cultivations of Corynebacterium glutamicum in a standard two-compartment reactor (two-CR) is compared to the performance in a novel three-compartment reactor (three-CR). The three-CR is designed to mimic three distinct zones of an industrial scale bioreactor that occur if the feed addition is installed at the bottom of the fluid phase. Our findings show that lactate and succinate appear in concentrations two-fold higher in the three-CR cultivation than in the two-CR cultivation. Similar results are revealed for the amino acids glycine, threonine, glutamate, and glutamine. In contrast to the two-CR cultivation, no intracellular accumulation of pyruvate is observed in the three-CR cultivation, since the carbon fluxes are directed toward lactate. As previously reported, the expression of lactate dehydrogenase (LDH) is increased in the context of oxygen deprivation. Thus, C. glutamicum adapts to the oscillating environment in the three-CR. This successful adaptation is revealed by a flow cytometric analysis of BOX-stained cells and a series of electrooptical at line measurements of cell polarisability. Both methods indicate a higher polarisability of cells in the three-CR cultivation. PI-staining does not indicate any membrane damage or accelerated cell death in either system. However, although the strain shows robustness, the product yield of lysine is reduced in scale-down cultivations as compared to cultivations at homogeneous conditions, which underlines the relevance of process optimization. PMID:25728062

  6. 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

  7. F-Actin-Dependent Endocytosis of Cell Wall Pectins in Meristematic Root Cells. Insights from Brefeldin A-Induced Compartments1

    PubMed Central

    Baluška, František; Hlavacka, Andrej; Šamaj, Jozef; Palme, Klaus; Robinson, David G.; Matoh, Toru; McCurdy, David W.; Menzel, Diedrik; Volkmann, Dieter

    2002-01-01

    Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1→4)-β-d-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner. PMID:12226521

  8. Characterization of a Novel Prevacuolar Compartment in Neurospora crassa.

    PubMed

    Bowman, Barry J; Draskovic, Marija; Schnittker, Robert R; El-Mellouki, Tarik; Plamann, Michael D; Sánchez-León, Eddy; Riquelme, Meritxell; Bowman, Emma Jean

    2015-12-01

    Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts. PMID:26453652

  9. Dopamine modulated ionic permeability in mesoporous silica sphere based biomimetic compartment.

    PubMed

    Liu, Wei; Yang, Xiaohai; He, Dinggeng; He, Leiliang; Li, Li; Liu, Yu; Liu, Jianbo; Wang, Kemin

    2016-06-01

    The building of artificial systems with similar structure and function as cellular compartments will expand our understanding of compartmentalization related biological process and facilitate the construction of biomimetic highly functional structures. Herein, surface phenylboronic acid functionalized mesoporous silica sphere was developed as a biomimetic dopamine gated compartment, in which the ionic permeability can be well modulated through the dopamine-binding induced charge reversal. As the phenylboronic acid is negatively charged, the negatively charged 1, 3, 6, 8-pyrenetetrasulfonic acid (TPSA) was hindered from permeation into the biomimetic compartment. However, the presence of dopamine and its binding with phenylboronic acid reversed the gatekeeper shell from negative to positive charged and gated the permeation of TPSA into the interior. The dopamine gated permeation phenomenon resembles that in biological system, and thus the phenylboronic acid functionalized mesoporous silica sphere was taken as a simple model for dopamine gated ion channel decorated biological compartment. It will also contribute to the development of artificial cell and responsive nanoreactor. PMID:26962763

  10. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse

    PubMed Central

    Singh, Rajesh K.; Barbosa-Lorenzi, Valéria C.; Lund, Frederik W.; Grosheva, Inna; Maxfield, Frederick R.; Haka, Abigail S.

    2016-01-01

    ABSTRACT Monocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub-regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane. PMID:26801085

  11. 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

  12. Models to predict unbound intracellular drug concentrations in the presence of transporters.

    PubMed

    Korzekwa, Ken R; Nagar, Swati; Tucker, Jalia; Weiskircher, Erica A; Bhoopathy, Siddhartha; Hidalgo, Ismael J

    2012-05-01

    Knowledge of free drug intracellular concentration is necessary to predict the impacts of drugs on intracellular targets. The goal of this study was to develop models to predict free intracellular drug concentrations in the presence of apical efflux transporters. The apical efflux transporter P-glycoprotein (P-gp), encoded by human gene multidrug resistance 1 (MDR1), was studied. Apparent permeabilities for 10 compounds in Madin-Darby canine kidney (MDCK) and MDR1-MDCK cell monolayers were obtained experimentally. Six of these compounds were evaluated additionally in the presence of the P-gp inhibitor cyclosporine A. A three-compartment model was developed, and passive and apical efflux clearances (CL(d) and CL(ae), respectively) were estimated. Endogenous canine transporters also were delineated. The three-compartment model was unable to simulate experimentally observed lag times and exhibited systematic bias across the simulations. Next, a five-compartment model with explicit membrane compartments was developed. This model resulted in lower systematic errors and simulated the lag time observed experimentally. Apical efflux was modeled out of the cell or out of the membrane. The five-compartment model with apical efflux out of the membrane predicted marked differences in unbound intracellular concentrations between the apical-to-basolateral and the basolateral-to-apical directions. Upon apical drug addition, large decreases in intracellular concentrations were observed with the efflux transporter. No such difference was predicted upon basolateral drug addition. This is consistent with experimental differences in the impact of P-gp on hepatic and brain distribution and supports the hypothesis that apical efflux occurs out of the apical membrane. PMID:22279052

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Delayed Presentation of Acute Gluteal Compartment Syndrome.

    PubMed

    Tasch, James J; Misodi, Emmanuel O

    2016-01-01

    BACKGROUND Acute gluteal compartment syndrome is a rare condition that usually results from prolonged immobilization following a traumatic event, conventionally involving the presence of compounding factors such as alcohol or opioid intoxication. If delay in medical treatment is prolonged, severe rhabdomyolysis may ensue, leading to acute renal failure and potentially death. CASE REPORT We report the case of a 23-year-old male with a recent history of incarceration and recreational drug use, who presented with reports of severe right-sided buttock pain and profound right-sided neurological loss following a questionable history involving prolonged immobilization after a fall from a standing position. The patient required an emergent gluteal fasciotomy immediately upon admission and required temporary hemodialysis. After an extended hospital stay, he ultimately recovered with only mild deficits in muscular strength in the right lower extremity. CONCLUSIONS This report demonstrates the importance of early recognition of gluteal compartment syndrome to prevent morbidity and mortality. Compartment syndrome presents in many unique ways, and healthcare practitioners must have a keen diagnostic sense to allow for early surgical intervention. Proper wick catheter measurements should be utilized more frequently, instead of relying on clinical symptomatology such as loss of peripheral pulses for diagnosis of compartment syndrome. PMID:27432320

  18. Gluteal Compartment Syndrome Secondary to Pelvic Trauma.

    PubMed

    Diaz Dilernia, Fernando; Zaidenberg, Ezequiel E; Gamsie, Sebastian; Taype Zamboni, Danilo E R; Carabelli, Guido S; Barla, Jorge D; Sancineto, Carlos F

    2016-01-01

    Gluteal compartment syndrome (GCS) is extremely rare when compared to compartment syndrome in other anatomical regions, such as the forearm or the lower leg. It usually occurs in drug users following prolonged immobilization due to loss of consciousness. Another possible cause is trauma, which is rare and has only few reports in the literature. Physical examination may show tense and swollen buttocks and severe pain caused by passive range of motion. We present the case of a 70-year-old man who developed GCS after prolonged anterior-posterior pelvis compression. The physical examination revealed swelling, scrotal hematoma, and left ankle extension weakness. An unstable pelvic ring injury was diagnosed and the patient was taken to surgery. Measurement of the intracompartmental pressure was measured in the operating room, thereby confirming the diagnosis. Emergent fasciotomy was performed to decompress the three affected compartments. Trauma surgeons must be aware of the possibility of gluteal compartment syndrome in patients who have an acute pelvic trauma with buttock swelling and excessive pain of the gluteal region. Any delay in diagnosis or treatment can be devastating, causing permanent disability, irreversible loss of gluteal muscles, sciatic nerve palsy, kidney failure, or even death. PMID:27579205

  19. 36 CFR 1192.127 - Sleeping compartments.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Sleeping compartments. 1192.127 Section 1192.127 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION VEHICLES Intercity Rail Cars and Systems §...

  20. What's new in acute compartment syndrome?

    PubMed

    Harvey, Edward J; Sanders, David W; Shuler, Michael S; Lawendy, Abdel-Rahman; Cole, Ashley L; Alqahtani, Saad M; Schmidt, Andrew H

    2012-12-01

    Acute compartment syndrome (ACS) after trauma is often the result of increased size of the damaged tissues after acute crush injury or from reperfusion of ischemic areas. It usually is not solely caused by accumulation of free blood or fluid in the compartment, although that can contribute in some cases. There is no reliable and reproducible test that confirms the diagnosis of ACS. A missed diagnosis or failure to cut the fascia to release pressure within a few hours can result in severe intractable pain, paralysis, and sensory deficits. Reduced blood circulation leads to oxygen and nutrient deprivation, muscle necrosis, and permanent disability. Currently, the diagnosis of ACS is made on the basis of physical examination and repeated needle sticks over a short time frame to measure intracompartmental pressures. Missed compartment syndromes continue to be one of most common causes of malpractice lawsuits. Existing technology for continuous pressure measurements are insensitive, particularly in the deep tissues and compartments, and their use is restricted to highly trained personnel. Newer concepts of the pathophysiology accompanied by new diagnostic and therapeutic modalities have recently been advanced. Among these are the concept of inflammatory mediators as markers and anti-inflammatories as medical adjunct therapy. New diagnostic modalities include near-infrared spectroscopy, ultrafiltration catheters, and radio-frequency identification implants. These all address current shortcomings in the diagnostic armamentarium that trauma surgeons can use. The strengths and weaknesses of these new concepts are discussed to allow the trauma surgeon to follow current evolution of the field. PMID:22913965

  1. Gluteal Compartment Syndrome Secondary to Pelvic Trauma

    PubMed Central

    Taype Zamboni, Danilo E. R.; Carabelli, Guido S.; Barla, Jorge D.; Sancineto, Carlos F.

    2016-01-01

    Gluteal compartment syndrome (GCS) is extremely rare when compared to compartment syndrome in other anatomical regions, such as the forearm or the lower leg. It usually occurs in drug users following prolonged immobilization due to loss of consciousness. Another possible cause is trauma, which is rare and has only few reports in the literature. Physical examination may show tense and swollen buttocks and severe pain caused by passive range of motion. We present the case of a 70-year-old man who developed GCS after prolonged anterior-posterior pelvis compression. The physical examination revealed swelling, scrotal hematoma, and left ankle extension weakness. An unstable pelvic ring injury was diagnosed and the patient was taken to surgery. Measurement of the intracompartmental pressure was measured in the operating room, thereby confirming the diagnosis. Emergent fasciotomy was performed to decompress the three affected compartments. Trauma surgeons must be aware of the possibility of gluteal compartment syndrome in patients who have an acute pelvic trauma with buttock swelling and excessive pain of the gluteal region. Any delay in diagnosis or treatment can be devastating, causing permanent disability, irreversible loss of gluteal muscles, sciatic nerve palsy, kidney failure, or even death. PMID:27579205

  2. 36 CFR 1192.127 - Sleeping compartments.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 36 Parks, Forests, and Public Property 3 2014-07-01 2014-07-01 false Sleeping compartments. 1192.127 Section 1192.127 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION...

  3. 36 CFR 1192.127 - Sleeping compartments.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 3 2011-07-01 2011-07-01 false Sleeping compartments. 1192.127 Section 1192.127 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION...

  4. 36 CFR 1192.127 - Sleeping compartments.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 36 Parks, Forests, and Public Property 3 2012-07-01 2012-07-01 false Sleeping compartments. 1192.127 Section 1192.127 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION...

  5. 36 CFR 1192.127 - Sleeping compartments.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 36 Parks, Forests, and Public Property 3 2013-07-01 2012-07-01 true Sleeping compartments. 1192.127 Section 1192.127 Parks, Forests, and Public Property ARCHITECTURAL AND TRANSPORTATION BARRIERS COMPLIANCE BOARD AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY GUIDELINES FOR TRANSPORTATION...

  6. Acute bilateral spontaneous forearm compartment syndrome.

    PubMed

    Dalton, David M; Munigangaiah, Sudarshan; Subramaniam, Tava; McCabe, John P

    2014-01-01

    Acute spontaneous compartment syndrome of the forearm is rarely reported in the literature. It is typically associated with trauma or thromboembolism in the acute setting and repetitive exertional stress in the chronic setting. However it is rare for it to present bilaterally with no apparent underlying cause. We report the case of a young 31-year-old lady who presented to our Emergency Department with bilateral compartment syndrome of the forearm. Her presenting complaints included acute severe pain and swelling of the forearms bilaterally, with a decreased range of movement of the wrist and fingers. She also complained of numbness in all fingers. She had no history of recent trauma and ultrasound scans showed no evidence of vascular compromise. Past medical history was notable only for idiopathic hypertension and coeliac disease. The patient was taken to theatre urgently where flexor and extensor compartments and carpal tunnel were decompressed. Pronator Teres was found to be dusky initially but turned pink after decompression. All other muscles were normal. An interesting fact of this case was that combination of the high compartment pressures and anaesthetic related hypotension caused the forearm pulses to become impalpable at induction, these returned intra-operatively. The patient has been seen in the outpatient department following discharge. She is well apart from some mildly reduced grip strength in her right hand likely due to carpal tunnel decompression. No cause was found for the scenario after extensive medical investigation. PMID:24641749

  7. Compartment Syndrome Resulting from Carbon Monoxide Poisoning.

    PubMed

    Serbest, Sancar; Belhan, Oktay; Gürger, Murat; Tosun, Haci Bayram

    2015-12-01

    Every year, especially in the cooler Fall and Winter months, hundreds of people die because of carbon monoxide poisoning. This occurs usually as an accident. It is a significant cause of poisoning worldwide. We present a case of compartment syndrome in both lower extremities with accompanying acute renal failure and systemic capillary leakage syndrome because of carbon monoxide poisoning. PMID:26588033

  8. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    PubMed Central

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  9. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

    PubMed

    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  10. CD22 is a recycling receptor that can shuttle cargo between the cell surface and endosomal compartments of B cells

    PubMed Central

    O‘Reilly, Mary K.; Tian, Hua; Paulson, James C.

    2010-01-01

    CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family that is known to be a regulator of B cell signaling. Its B cell-specific expression makes it an attractive target for immunotoxin-mediated B cell depletion therapy for the treatment of B cell lymphomas and autoimmune diseases. Although CD22 is well documented to be an endocytic receptor, it is believed that following internalization it is targeted for degradation. We show here that CD22 is instead constitutively recycled to the cell surface. We also find that glycan ligand–based cargo is released from CD22 and accumulates intracellularly as CD22 recycles between the cell surface and endosomal compartments. In contrast, antibodies to CD22 do not accumulate, but remain bound to CD22 and recycle to the cell surface. The results have implications for development of agents that target CD22 as an endocytic receptor for delivery of cytotoxic cargo to B cells. PMID:21178016

  11. SERS nanosensors that report pH of endocytic compartments during FcεRI transit

    PubMed Central

    Nowak-Lovato, K.L.; Wilson, Bridget S.; Rector, K.D.

    2011-01-01

    Recently, the development of an IgE receptor (FcεRI)-targeted, pH sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated [1]. The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37°C) versus room temperature (25°C). As expected, we observed markedly slower progression of receptors through low pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H+ ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na+/H+ exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15–20 minutes to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors. PMID:20842349

  12. Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    PubMed Central

    Repella, Tana L.; Ho, Mengfei; Chong, Tracy P. M.; Bannai, Yuka; Wilson, Brenda A.

    2011-01-01

    The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity. PMID:22053287

  13. Exertional Compartment Syndrome of the Medial Foot Compartment: Diagnosis and Treatment.

    PubMed

    Izadi, Faye E; Richie Jr, Douglas H

    2014-06-25

    Abstract Exertional compartment syndrome (ECS) in the foot is rarely reported and often confused with plantar fasciitis as a cause of arch pain in the running athlete. We describe a case involving a 19 year old competitive collegiate runner who developed a chronic case of bilateral medial arch pain during training, which was initially diagnosed as plantar fasciitis but failed to respond to conventional treatment. After symptoms began to suggest exertional compartment syndrome, the diagnosis was confirmed by measuring an elevated resting pressure in the medial compartment of both feet. The patient underwent a bilateral medial compartment fasciotomy, which allowed a full return to activity, and has remained pain free after a one year follow up. PMID:24963970

  14. Exertional compartment syndrome of the medial foot compartment--diagnosis and treatment: a case report.

    PubMed

    Izadi, Faye E; Richie, Douglas H

    2014-07-01

    Exertional compartment syndrome in the foot is rarely reported and often confused with plantar fasciitis as a cause of arch pain in the running athlete. We describe a case involving a 19-year-old competitive collegiate runner who developed a chronic case of bilateral medial arch pain during training, which was initially diagnosed as plantar fasciitis but failed to respond to conventional treatment. After symptoms began to suggest exertional compartment syndrome, the diagnosis was confirmed by measuring an elevated resting pressure in the medial compartment of both feet. The patient underwent a bilateral medial compartment fasciotomy, which allowed a full return to activity, and has remained pain free after a 1-year follow-up. PMID:25076087

  15. 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

  16. 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

  17. 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

  18. 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

  19. Glutamate Utilization Couples Oxidative Stress Defense and the Tricarboxylic Acid Cycle in Francisella Phagosomal Escape

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Rigard, Mélanie; Dairou, Julien; Dupuis, Marion; Dubail, Iharilalao; Meibom, Karin; Henry, Thomas; Barel, Monique; Charbit, Alain

    2014-01-01

    Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle. PMID:24453979

  20. Dynamic GLUT4 sorting through a syntaxin-6 compartment in muscle cells is derailed by insulin resistance-causing ceramide

    PubMed Central

    Foley, Kevin P.; Klip, Amira

    2014-01-01

    ABSTRACT GLUT4 constitutively recycles between the plasma membrane and intracellular depots. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting GLUT4 to the plasma membrane from insulin-responsive vesicles. Muscle is the primary site for dietary glucose deposition; however, how GLUT4 sorts into insulin-responsive vesicles, and if and how insulin resistance affects this process, is unknown. In L6 myoblasts stably expressing myc-tagged GLUT4, we analyzed the intracellular itinerary of GLUT4 as it internalizes from the cell surface and examined if such sorting is perturbed by C2-ceramide, a lipid metabolite causing insulin resistance. Surface-labeled GLUT4myc that internalized for 30 min accumulated in a Syntaxin-6 (Stx6)- and Stx16-positive perinuclear sub-compartment devoid of furin or internalized transferrin, and displayed insulin-responsive re-exocytosis. C2-ceramide dispersed the Stx6-positive sub-compartment and prevented insulin-responsive re-exocytosis of internalized GLUT4myc, even under conditions not affecting insulin-stimulated signaling towards Akt. Microtubule disruption with nocodazole prevented pre-internalized GLUT4myc from reaching the Stx6-positive perinuclear sub-compartment and from undergoing insulin-responsive exocytosis. Removing nocodazole allowed both parameters to recover, suggesting that the Stx6-positive perinuclear sub-compartment was required for GLUT4 insulin-responsiveness. Accordingly, Stx6 knockdown inhibited by ∼50% the ability of internalized GLUT4myc to undergo insulin-responsive re-exocytosis without altering its overall perinuclear accumulation. We propose that Stx6 defines the insulin-responsive compartment in muscle cells. Our data are consistent with a model where ceramide could cause insulin resistance by altering intracellular GLUT4 sorting. PMID:24705014

  1. Secretory compartments as instances of dynamic self-evolving structures.

    PubMed

    Képès, François

    2002-01-01

    Biological objects are often "constructive dynamic systems" whose structures evolve as a consequence of their internal dynamics, which in turn is affected by the overall structure. As very few tools are presently adapted to tackle constructive dynamic systems, they constitute fascinating challenges for modeling/simulation. In cell biology, the secretory process in eukaryotic cells corresponds to this type of system, as it appears to autonomously generate new structures as a result of its molecular dynamics. Here I briefly review the only documented case of a membrane-bounded intracellular compartment whose very existence strictly depends on its continued functioning. Indeed, the Golgi apparatus of the yeast Saccharomyces cerevisiae appears at steady-state as a continuously renewed set of transitory membrane-bounded structures that self-mature, rather than as a permanent entity. On the basis of this case and of recent advances in related molecular studies, a detailed model is proposed, that encompasses the birth of a yeast Golgi element and bridges its molecular and morphogenetic aspects. This model is extended to briefly outline three evolutionary "inventions", from S. cerevisiae to another yeast, Pichia pastoris, on to plant, and on to animal cells: stacking, stabilizing and aggregating the primary Golgi elements. PMID:12675528

  2. Patterning and Compartment Formation in the Diencephalon

    PubMed Central

    Chatterjee, Mallika; Li, James Y. H.

    2012-01-01

    The diencephalon gives rise to structures that play an important role in connecting the anterior forebrain with the rest of the central nervous system. The thalamus is the major diencephalic derivative that functions as a relay station between the cortex and other lower order sensory systems. Almost two decades ago, neuromeric/prosomeric models were proposed describing the subdivision and potential segmentation of the diencephalon. Unlike the laminar structure of the cortex, the diencephalon is progressively divided into distinct functional compartments consisting principally of thalamus, epithalamus, pretectum, and hypothalamus. Neurons generated within these domains further aggregate to form clusters called nuclei, which form specific structural and functional units. We review the recent advances in understanding the genetic mechanisms that are involved in the patterning and compartment formation of the diencephalon. PMID:22593732

  3. A modern definition of mediastinal compartments.

    PubMed

    Carter, Brett W; Tomiyama, Noriyuki; Bhora, Faiz Y; Rosado de Christenson, Melissa L; Nakajima, Jun; Boiselle, Phillip M; Detterbeck, Frank C; Marom, Edith M

    2014-09-01

    Division of the mediastinum into compartments is used to help narrow the differential diagnosis of newly detected mediastinal masses, to assist in planning biopsy and surgical procedures, and to facilitate communication among clinicians of multiple disciplines. Several traditional mediastinal division schemes exist based upon arbitrary landmarks on the lateral chest radiograph. We describe a modern, computed tomography-based mediastinal division scheme, which has been accepted by the International Thymic Malignancy Interest Group as a new standard. This clinical classification defines a prevascular (anterior), a visceral (middle), and a paravertebral (posterior) compartment, with anatomic boundaries defined clearly by computed tomography. It is our intention that this definition be used in the reporting of clinical cases and the design of prospective clinical trials. PMID:25396318

  4. Monoterpene biosynthesis potential of plant subcellular compartments.

    PubMed

    Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. PMID:26356766

  5. [Acute compartment syndrome after a bowling game].

    PubMed

    Meyer, C Y; Braun, K F; Huber-Wagner, S; Neu, J

    2015-11-01

    A 28-year-old male patient was initially conservatively treated by a general physician for muscle strain of the right calf after a bowling game. Due to increasing pain and swelling of the lower leg 5 days later, the differential diagnosis of a deep vein thrombosis was considered. Furthermore, the onset of neurological deficits and problems with raising the foot prompted inclusion of compartment syndrome in the differential diagnosis for the first time. Admission to hospital for surgical intervention was scheduled for the following day. At this point in time the laboratory results showed a negative d-dimer value and greatly increased C-reactive protein level. On day 6 a dermatofasciotomy was performed which revealed extensive muscular necrosis with complete palsy of the peroneal nerve. In the following lawsuit the patient accused the surgeon of having misdiagnosed the slow-onset compartment syndrome and thus delaying correct and mandatory treatment. The arbitration board ruled that the surgeon should have performed fasciotomy immediately on day 5 at the patient's consultation. The clinical presentation of progressive pain, swelling of the lower leg in combination with peroneal palsy must lead to the differential diagnosis of compartment syndrome resulting in adequate therapy. The delay of immediate surgery, therefore, was assessed to be faulty as this knowledge is to be expected of a surgeon. PMID:26440405

  6. [Arthritis of the Medial Knee Joint Compartment].

    PubMed

    Matziolis, G; Röhner, E

    2015-10-01

    23 % of all persons older than 65 years suffer from osteoarthritis of the medial compartment of the knee joint, a very common situation in orthopaedic practice 1. As a result of the demographic trend the number of patients is expected to increase in the future. Based on specific joint biomechanics and kinematics the medial knee joint compartment is more frequently affected than the lateral. Only an understanding of the functional anatomy and underlying pathology allows a critical evaluation of different available conservative and operative treatment options. This article gives an overview of diagnostic and therapeutic strategies of osteoarthritis of the medial knee joint. Frequently performed surgeries, e.g. high tibial osteotomy (HTO), unicompartmental knee arthroplasty (UKA) and total knee arthroplasty (TKA) will be presented in a comparative manner. The actual scientific evidence will be given with the goal of an evidence based therapy that is adopted to stage and pathology of osteoarthritis of the medial compartment of the knee joint. PMID:26451864

  7. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    PubMed

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied. PMID:26795486

  8. 14 CFR 91.613 - Materials for compartment interiors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... compartment interiors. (a) No person may operate an airplane that conforms to an amended or supplemental type... SFAR the airplane meets the compartment interior requirements set forth in § 25.853 (a), (b), (b-1),...

  9. 14 CFR 25.855 - Cargo or baggage compartments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... quantities of smoke or extinguishing agent into compartments occupied by the crew or passengers, and (3) The... that no inadvertent operation of smoke or fire detectors in any compartment would occur as a result...

  10. 14 CFR 25.855 - Cargo or baggage compartments.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... compartments, as defined in § 25.857, must have a liner, and the liner must be separate from (but may be attached to) the airplane structure. (c) Ceiling and sidewall liner panels of Class C compartments...

  11. 14 CFR 25.855 - Cargo or baggage compartments.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... compartments, as defined in § 25.857, must have a liner, and the liner must be separate from (but may be attached to) the airplane structure. (c) Ceiling and sidewall liner panels of Class C compartments...

  12. 14 CFR 25.855 - Cargo or baggage compartments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... compartments, as defined in § 25.857, must have a liner, and the liner must be separate from (but may be attached to) the airplane structure. (c) Ceiling and sidewall liner panels of Class C compartments...

  13. 14 CFR 25.855 - Cargo or baggage compartments.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... compartments, as defined in § 25.857, must have a liner, and the liner must be separate from (but may be attached to) the airplane structure. (c) Ceiling and sidewall liner panels of Class C compartments...

  14. 46 CFR 174.075 - Compartments assumed flooded: general.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Units § 174.075 Compartments assumed flooded: general. The individual flooding of each of the... § 174.065 (a). Simultaneous flooding of more than one compartment must be assumed only when indicated...

  15. 46 CFR 174.075 - Compartments assumed flooded: general.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Units § 174.075 Compartments assumed flooded: general. The individual flooding of each of the... § 174.065 (a). Simultaneous flooding of more than one compartment must be assumed only when indicated...

  16. 46 CFR 174.075 - Compartments assumed flooded: general.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Units § 174.075 Compartments assumed flooded: general. The individual flooding of each of the... § 174.065 (a). Simultaneous flooding of more than one compartment must be assumed only when indicated...

  17. 46 CFR 174.075 - Compartments assumed flooded: general.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Units § 174.075 Compartments assumed flooded: general. The individual flooding of each of the... § 174.065 (a). Simultaneous flooding of more than one compartment must be assumed only when indicated...

  18. 46 CFR 174.075 - Compartments assumed flooded: general.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Units § 174.075 Compartments assumed flooded: general. The individual flooding of each of the... § 174.065 (a). Simultaneous flooding of more than one compartment must be assumed only when indicated...

  19. [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

  20. Compartment Syndrome of the Hand: A Little Thought about Diagnosis

    PubMed Central

    Reichman, Eric F.

    2016-01-01

    Compartment syndrome of the forearm is a well described entity but there have been relatively few case reports in the emergency medicine literature of hand compartment syndromes (HCS). Prompt recognition and treatment of this potential limb threat are essential to minimize morbidity and mortality. Presented is a case of a documented hand compartment syndrome following a motor vehicle collision. PMID:27293917

  1. 14 CFR 29.1193 - Cowling and engine compartment covering.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Cowling and engine compartment covering. 29... Protection § 29.1193 Cowling and engine compartment covering. (a) Each cowling and engine compartment... of § 29.1187. (c) On rotorcraft with a diaphragm isolating the engine power section from the...

  2. 14 CFR 27.773 - Pilot compartment view.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment view. 27.773 Section 27... § 27.773 Pilot compartment view. (a) Each pilot compartment must be free from glare and reflections that could interfere with the pilot's view, and designed so that— (1) Each pilot's view is...

  3. 14 CFR 29.773 - Pilot compartment view.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment view. 29.773 Section 29... Accommodations § 29.773 Pilot compartment view. (a) Nonprecipitation conditions. For nonprecipitation conditions, the following apply: (1) Each pilot compartment must be arranged to give the pilots a...

  4. 14 CFR 25.857 - Cargo compartment classification.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Cargo compartment classification. 25.857....857 Cargo compartment classification. (a) Class A; A Class A cargo or baggage compartment is one in... detector or fire detector system to give warning at the pilot or flight engineer station. (c) Class C....

  5. 14 CFR 29.773 - Pilot compartment view.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment view. 29.773 Section 29... Accommodations § 29.773 Pilot compartment view. (a) Nonprecipitation conditions. For nonprecipitation conditions, the following apply: (1) Each pilot compartment must be arranged to give the pilots a...

  6. 14 CFR 23.773 - Pilot compartment view.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment view. 23.773 Section 23... Personnel and Cargo Accommodations § 23.773 Pilot compartment view. (a) Each pilot compartment must be— (1) Arranged with sufficiently extensive, clear and undistorted view to enable the pilot to safely...

  7. 14 CFR 27.773 - Pilot compartment view.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment view. 27.773 Section 27... § 27.773 Pilot compartment view. (a) Each pilot compartment must be free from glare and reflections that could interfere with the pilot's view, and designed so that— (1) Each pilot's view is...

  8. 14 CFR 23.773 - Pilot compartment view.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment view. 23.773 Section 23... Personnel and Cargo Accommodations § 23.773 Pilot compartment view. (a) Each pilot compartment must be— (1) Arranged with sufficiently extensive, clear and undistorted view to enable the pilot to safely...

  9. Chronic exertional compartment syndrome in adductor pollicis muscle: case report.

    PubMed

    Lee, Chang-Hun; Lee, Kwang-Hyun; Lee, Seung-Hun; Kim, Yee-Suk; Chung, Ung-Seo

    2012-11-01

    We report a case of chronic exertional compartment syndrome in the adductor pollicis that was confirmed by measuring elevated compartment pressure. Specific finding of magnetic resonance imaging, increased T2 signal intensity in the involved compartment, was also useful for the diagnosis. Pain was relieved by fasciotomy through a volar approach. PMID:23040640

  10. Two-compartment behavior during transport of folate compounds in L1210 cell plasma membrane vesicles

    SciTech Connect

    Yang, C.H.; Dembo, M.; Sirotnak, F.M.

    1982-01-01

    The transport of (/sup 3/H) 1,L 5-formyltetrahydrofolate, (/sup 3/H) folic acid, and (/sup 3/H)methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport.

  11. 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

  12. 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

  13. The cooperative electrochemical oxidation of chlorophenols in anode-cathode compartments.

    PubMed

    Wang, Hui; Wang, Jian Long

    2008-06-15

    By using a self-made carbon/polytetrafluoroethylene (C/PTFE) O2-fed as the cathode and Ti/IrO2/RuO2 as the anode, the degradation of three organic compounds (phenol, 4-chlorophenol, and 2,4-dichlorophenol) was investigated in the diaphragm (with terylene as diaphragm material) electrolysis device by electrochemical oxidation process. The result indicated that the concentration of hydrogen peroxide (H2O2) was 8.3 mg/L, and hydroxyl radical (HO) was determined in the cathodic compartment by electron spin resonance spectrum (ESR). The removal efficiency for organic compounds reached about 90% after 120 min, conforming to the sequence of phenol, 4-chlorophenol, and 2,4-dichlorophenol. And the dechlorination degree of 4-chlorophenol exceeded 90% after 80 min. For H2O2, HO existed in the catholyte and reduction dechlorination at the cathode, the mineralization of organics in the cathodic compartment was better than that in the anodic compartment. The degradation of organics was supposed to be cooperative oxidation by direct or indirect electrochemical oxidation at the anode and H2O2, HO produced by oxygen reduction at the cathode. High-performance liquid chromatography (HPLC) allowed identifying phenol as the dechlorination product of 4-chlorophenol in the cathodic compartment, and hydroquinone, 4-chlorocatechol, benzoquinone, maleic, fumaric, oxalic, and formic acids as the main oxidation intermediates in the cathodic and anodic compartments. A reaction scheme involving all these intermediates was proposed. PMID:17996367

  14. Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells.

    PubMed

    Abrahamse, S L; Pool-Zobel, B L; Rechkemmer, G

    1999-04-01

    Short chain fatty acids (SCFA) are considered to be beneficial fermentation products in the gut by exerting trophic effects in non-transformed colon cells and by slowing proliferation and enhancing differentiation in colonic tumour cells. We have studied the further effects of SCFA on cellular events of early carcinogenesis, genotoxicity and cytotoxicity in rat distal colon cells. Cytotoxicity was assessed by measuring trypan blue exclusion and by determining the H2O2-induced changes in intracellular calcium concentration ([Ca2+]i) using a fluorospectrophotometer and the calcium-sensitive fluorescent dye Fura-2. The microgel electrophoresis technique (COMET assay) was used to assess oxidative DNA damage. Individual SCFA and physiological SCFA mixtures were investigated for their potential to prevent DNA and cell damage induced by H2O2. For this, freshly isolated colon cells were treated with H2O2 (100-500 microM) and 6.25 mM SCFA. We have found 100-500 microM H2O2 to cause a fast initial increase in [Ca2+]i, whereafter the levels gradually further increased. Addition of SCFA did not affect [Ca2+]i nor did it reduce the H2O2-induced increase in [Ca2+]i. Butyrate and acetate were able to reduce the induction of DNA damage by 100, 200 and 500 microM H2O2, respectively. In contrast, i-butyrate and propionate were ineffective. The degree of reduction of DNA damage for the two protective SCFA was similar. Physiological mixtures containing acetate, propionate and butyrate in ratios of 41:21:38 or 75:15:10 that are expected to arise in the colon after fermentation of resistant starches and pectin, respectively, did not show significant antigenotoxic effects. The major difference between butyrate and acetate, on one hand, and i-butyrate and propionate, on the other hand, is that the former compounds are utilized best as energy sources by the colon cells. Therefore, our results on antigenotoxicity coupled with the findings on [Ca2+]i homeostasis indicate that molecular

  15. 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

  16. Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

    PubMed Central

    Schmidt, W K; Moore, H P

    1995-01-01

    Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing. Images PMID:8573786

  17. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

    PubMed

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms. PMID:27055246

  18. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation

    PubMed Central

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G. John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg’) shows that cells with the lowest value of intracellular Tg’ survive the freezing process better than cells with a higher intracellular Tg’. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms. PMID:27055246

  19. Orbital compartment syndrome following aneurysm surgery.

    PubMed

    Gauden, Andrew J; Hardy, Thomas; Mack, Heather G; Danesh-Meyer, Helen V; Kaye, Andrew H

    2012-07-01

    Orbital compartment syndrome (OCS) is a rare cause of blindness following intracranial surgery. We report a patient with OCS following intracranial cerebrovascular surgery precipitated by severe straining. OCS occurred due to a rapid increase in intraorbital pressure within the rigid confines of the orbit causing hypoperfusion of critical neural structures, which resulted in visual loss and a complete external ophthalmoplegia. Treatment involved urgent surgical soft tissue decompression of the orbit, corticosteroids and osmotic agents. It is important to consider OCS as a cause of blindness in the neurosurgical postoperative setting as without rapid treatment this condition has a very poor prognosis. PMID:22555128

  20. Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes.

    PubMed

    Fernández-Arenas, Elena; Bleck, Christopher K E; Nombela, César; Gil, Concha; Griffiths, Gareth; Diez-Orejas, Rosalía

    2009-04-01

    The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO. PMID:19134116

  1. Intracellular Toll-like receptor recruitment and cleavage in endosomal/lysosomal organelles.

    PubMed

    Tohmé, Mira; Manoury, Bénédicte

    2014-01-01

    Microbial pathogens are recognized through multiple, distinct receptors such as intracellular Toll-like receptors (TLRs 3, 7, 8, 9, and 13) which reside in the endosomes and lysosomes. TLRs are sensitive to chloroquine, a lysomotropic agent that neutralizes acidic compartments indicating a role for endo/lysosomal proteases for their signaling. Indeed, upon stimulation, full-length TLR7 and 9 are cleaved into a C-terminal fragment and this processing is highly dependent on a cysteine protease named asparagine endopeptidase (AEP) in dendritic cells. A recruitment and a boost in AEP activity, which was induced shortly after TLR7 and 9 stimulation, are shown to promote TLR7 and 9 cleavage and correlate with an increased acidification in endosomes and lysosomes. Moreover, mutating a putative AEP cleavage site in TLR7 or 9 strongly decreases their signaling in DCs, suggesting perhaps a direct cleavage of TLR7 and 9 by AEP. These results demonstrate that TLR7 and 9 require a proteolytic cleavage for their signaling and identified a key endocytic protease playing a critical role in this process. PMID:24377922

  2. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  3. Ultrasonic Apparatus and Method to Assess Compartment Syndrome

    NASA Technical Reports Server (NTRS)

    Yost, William T. (Inventor); Ueno, Toshiaki (Inventor); Hargens, Alan R. (Inventor)

    2009-01-01

    A process and apparatus for measuring pressure buildup in a body compartment that encases muscular tissue. The method includes assessing the body compartment configuration and identifying the effect of pulsatible components on compartment dimensions and muscle tissue characteristics. This process is used in preventing tissue necrosis, and in decisions of whether to perform surgery on the body compartment for prevention of Compartment Syndrome. An apparatus is used for measuring pressure build-up in the body compartment having components for imparting ultrasonic waves such as a transducer, placing the transducer to impart the ultrasonic waves, capturing the imparted ultrasonic waves, mathematically manipulating the captured ultrasonic waves and categorizing pressure build-up in the body compartment from the mathematical manipulations.

  4. Synthesis, Antimalarial Activity, and Intracellular Targets of MEFAS, a New Hybrid Compound Derived from Mefloquine and Artesunate▿

    PubMed Central

    de Pilla Varotti, Fernando; Botelho, Ana Cristina C.; Andrade, Anderson Assunção; de Paula, Renata C.; Fagundes, Elaine M. S.; Valverde, Alessandra; Mayer, Lúcia M. U.; Mendonça, Jorge Souza; de Souza, Marcus V. N.; Boechat, Núbia; Krettli, Antoniana Ursine

    2008-01-01

    A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca2+ at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H+ pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria. PMID

  5. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    PubMed Central

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  6. Changes in body fluid compartments during a 28-day bed rest

    NASA Technical Reports Server (NTRS)

    Fortney, Suzanne M.; Hyatt, Kenneth H.; Davis, John E.; Vogel, John M.

    1991-01-01

    Serial isotope measurements were used to obtain measurements of the body fluid responses of 10 22-29-year-old men during 28 d of simulated microgravity (bed rest). The subjects were maintained on a controlled metabolic diet for 7 d before the study, during 14 d of ambulatory control, 28 d of horizontal bed rest, and 14 d of ambulant recovery. Fluid compartments were measured on control days 1 and 9, bed rest days 2, 14, and 28, and recovery days 7 and 14. By day 2 of bed rest, plasma volume and extracellular volume (ECV) decreased significantly by an average 209 and 533 ml, respectively. Red cell volume and total body water (TBW) decreased more slowly, with average losses of 128 and 1316 ml, respectively, after 28 d of bed rest. Early in the bed rest, TBW loss was mostly from the ECV. Thereafter, the TBW deficit was derived from the intracellular compartment, which decreased an average of 838 ml after 28 d. These results suggest losses from all fluid compartments during bed rest, with no evidence of restoration of ECV after 1-2 weeks.

  7. Gluteal compartment syndrome after prolonged immobilisation.

    PubMed

    Liu, H L; Wong, David S Y

    2009-04-01

    Muscles in the gluteal region are confined by distinct fascial attachments which can potentially result in compartment syndrome. A 74-year-old chronic drinker was admitted to the medical ward after being found drunk on the street. He noticed acute painful swelling of the right side of his buttock the following morning and recalled a slip and fall prior to his blackout. The whole right half of the buttock was tense with erythematous overlying skin. Examination revealed sciatic nerve palsy and myoglobinuria. Emergency fasciotomy and debridement were performed. Intra-operative pressure measurement confirmed a grossly elevated intra-compartmental pressure. Gluteal compartment syndrome is an extremely rare condition and has only been scantily documented previously in case reports. Early diagnosis is crucial but delay recognition is common from lack of knowledge of the condition and readily results in permanent sciatic nerve injury and acute renal shutdown from myoglobinuria. Awareness of the condition, early diagnosis and prompt exploration provide the only chance of avoiding these devastating consequences. Acute swelling diffusely affecting the whole or one side of the buttock, a history of trauma and prolonged local pressure impingement associated with pain out of proportion to the clinical signs should raise a suspicion of this rare condition. PMID:19423461

  8. Remote detection of pressure compartments. Topical report

    SciTech Connect

    Surdam, R.C.; Boyd, N.; Jiao, Z.; Maucione, D.; Kubicheck, S.

    1996-02-01

    A significant portion of the Cretaceous shale section in the Rocky Mountain Laramide Basins (RMLB) is anomalously pressured and gas saturated. The top of the anomalously pressured zone is identified by marked increases in sonic transit time, hydrocarbon production index (P.I.), clay diagenesis (smectite to illite), and vitrinite reflectance gradients. The driving mechanism of anomalous pressure development and compartmentalization is the generation and storage of liquid hydrocarbons that subsequently partially react to gas, converting the fluid-flow system to a multiphase regime in which capillarity controls permeability; the result is elevated displacement pressure within the shales. Sandstone reservoirs within this anomalously pressured shale section are subdivided stratigraphically and diagenetically into relatively small, isolated pressure or fluid-flow compartments. The saturation of these compartments with hydrocarbons and the subsequent oil-to-gas reaction causes explusion of a significant portion of the free water, resulting in anomalously pressured gas accumulations characterized by depletion drive. The determination of the position and configuration of the pressure boundary between normal and anomalously pressured regimes and the detection and delineation of porosity/permeability `sweet spots` below this boundary are the two most important elements in exploring for basin center gas in the RMLB.

  9. Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors.

    PubMed Central

    Eskild, W; Kindberg, G M; Smedsrod, B; Blomhoff, R; Norum, K R; Berg, T

    1989-01-01

    Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial

  10. In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.

    PubMed

    Orij, Rick; Postmus, Jarne; Ter Beek, Alex; Brul, Stanley; Smits, Gertien J

    2009-01-01

    The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pHcyt) and mitochondrial pH (pHmit) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pHcyt of 7.2 and a constant pHmit of 7.5 in cells exponentially growing on glucose. We show that pHcyt and pHmit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pHi decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between pHi and growth. PMID:19118367

  11. 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...

  12. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. PMID:26195825

  13. 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

  14. The Orbital Workshop Waste Management Compartment

    NASA Technical Reports Server (NTRS)

    1972-01-01

    This image is a wide-angle view of the Orbital Workshop waste management compartment. The waste management facilities presented a unique challenge to spacecraft designers. In addition to collection of liquid and solid human wastes, there was a medical requirement to dry all solid human waste products and to return the residue to Earth for examination. Liquid human waste (urine) was frozen for return to Earth. Total quantities of each astronaut's liquid and solid wastes were precisely measured. Cabin air was drawn into the toilet, shown on the wall at right in this photograph, and over the waste products to generate a flow of the waste in the desired direction. The air was then filtered for odor control and antiseptic purposes prior to being discharged back into the cabin.

  15. The extracellular compartments of frog skeletal muscle.

    PubMed Central

    Neville, M C; Mathias, R T

    1979-01-01

    1. Detailed studies of solute efflux from frog sartorius muscle and single muscle fibres were carried out in order to characterize a 'special region' (Harris, 1963) in the extracellular space of muscle and determine whether this 'special region' is the sarcoplasmic reticulum. 2. The efflux of radioactive Na, Cl, glusose, 3-O-methylglucose, xylose, glycine, leucine, cycloleucine, Rb, K, inulin (mol. wt. 5000) and dextran (mol. wt. 17,000) from previously loaded muscles was studied. In all cases except dextran the curve had three components, a rapid (A) component which could be equated with efflux from the extracellular space proper, a slow (C) component representing cellular solute and an intermediate (B) component. The distribution space for the B component was 8% of muscle volume in summer frogs and 12% in winter frogs and appeared to be equal for all compounds studied. We tested the hypothesis that the B component originated from the sarcoplasmic reticulum. 3. The C component was missing from the dextran curves. Both dextran and inulin entered the compartment of origin of the B component (compartment B) to the same extent as small molecules. 4. For all compounds studies, the efflux rate constant for the A component could be predicted from the diffusion coefficient. For the B component the efflux rate constant was 6--10 times slower than that for the A component but was still proportional to the diffusion coefficient for the solute in question. 5. When Na and sucrose efflux from single fibres was followed, a B component was usually observed. The average distribution space for this component was small, averaging 1.5% of fibre volume. There was no difference between the average efflux rate constants for Na and sucrose. 6. In an appendix, the constraints placed on the properties of a hypothetical channel between the sarcoplasmic reticulum and the T-system by the linear electrical parameters of frog skeletal muscle are derived. It is shown that the conductance of such

  16. Dynamic Compartments in the Imperative π-Calculus

    NASA Astrophysics Data System (ADS)

    John, Mathias; Lhoussaine, Cédric; Niehren, Joachim

    Dynamic compartments with mutable configurations and variable volumes are of basic interest for the stochastic modeling of biochemistry in cells. We propose a new language to express dynamic compartments that we call the imperative π -calculus. It is obtained from the attributed π -calculus by adding imperative assignment operations to a global store. Previous approaches to dynamic compartments are improved in flexibility or efficiency. This is illustrated by an appropriate model of osmosis and a correct encoding of bioambBioAmbients.

  17. Vehicle hydraulic system that provides heat for passenger compartment

    DOEpatents

    Bartley, Bradley E.; Blass, James R.; Gibson, Dennis H.

    2001-01-01

    A vehicle includes a vehicle housing which defines a passenger compartment. Attached to the vehicle housing is a hydraulic system, that includes a hydraulic fluid which flows through at least one passageway within the hydraulic system. Also attached to the vehicle housing is a passenger compartment heating system. The passenger compartment heating system includes a heat exchanger, wherein a portion of the heat exchanger is a segment of the at least one passageway of the hydraulic system.

  18. 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

  19. Delayed onset thigh compartment syndrome secondary to contusion.

    PubMed

    Joglekar, Siddharth B; Rehman, Saqib

    2009-08-01

    While thigh compartment syndrome is relatively uncommon, it can occur in various situations. Multiple reports document thigh contusions as a cause of acute compartment syndrome; however, compartment syndrome of the thigh presenting primarily in a delayed fashion secondary to a contusion has not been described. This article reports a case of thigh compartment syndrome. A 39-year-old man sustained a left thigh contusion while playing basketball. He continued to play and also worked at the office over the next 2 days. Fifty-two hours postinjury, he developed severe pain in the thigh after a long walk. Increased swelling of the thigh followed, with numbness in the anterolateral thigh and pain with knee motion. He presented 60 hours postinjury with a compartment syndrome, and a lateral decompressive fasciotomy of the thigh was performed 62 hours postinjury. The wound was closed after 5 days. Three months postoperatively, the patient returned to playing basketball with no deficits. Treatment of established compartment syndrome in such cases is controversial, with some reports recommending nonoperative management. Contusion-related compartment syndromes are frequently associated with intramuscular bleeding in the involved compartment, which may accumulate slowly or worsen with further activity. Guidelines regarding return to sports need to be established in individuals sustaining severe contusions during sports-related activities to prevent compartment syndrome. Any individual sustaining such an injury should be under surveillance for delayed onset symptoms or signs of this potentially devastating syndrome. PMID:19708619

  20. Coping with the diagnostic complexities of the compartment syndrome

    NASA Technical Reports Server (NTRS)

    Mubarak, S. J.; Hargens, A. R.; Karkal, S. S.

    1988-01-01

    This review recognizes that, given the various complexities associated with the condition, no pat answers can be given to fit every patient with the compartment syndrome. The authors first give a definition of the syndrome, together with a brief account of how this self-perpetuating pathologic cycle is triggered. Next, they delineate specific anatomical features of compartments that are likely to be involved, and follow this with an inventory of symptoms and signs to look for in suspected cases. After sorting out the entities that can mimic the compartment syndrome, the authors describe three essential techniques of measuring tissue pressure, which can prove invaluable in diagnosing the compartment syndrome.

  1. 7 CFR 58.510 - Rooms and compartments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... compartments shall be ventilated to maintain sanitary conditions, preclude the growth of mold and air borne bacterial contaminants, prevent undue condensation of water vapor and minimize or eliminate...

  2. 7 CFR 58.510 - Rooms and compartments.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... compartments shall be ventilated to maintain sanitary conditions, preclude the growth of mold and air borne bacterial contaminants, prevent undue condensation of water vapor and minimize or eliminate...

  3. The MHC Class II-Associated Invariant Chain Interacts with the Neonatal Fcγ Receptor and Modulates Its Trafficking to Endosomal/Lysosomal Compartments1

    PubMed Central

    Ye, Lilin; Liu, Xindong; Rout, Subrat N.; Li, Zili; Yan, Yongqi; Lu, Li; Kamala, Tirumalai; Nanda, Navreet K.; Song, Wenxia; Samal, Siba K.; Zhu, Xiaoping

    2009-01-01

    The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-β2-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-γ-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-γ. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain. PMID:18684948

  4. Proton-assisted amino acid transporters are conserved regulators of proliferation and amino acid-dependent mTORC1 activation

    PubMed Central

    Heublein, S; Kazi, S; Ögmundsdóttir, M H; Attwood, E V; Kala, S; Boyd, C A R; Wilson, C; Goberdhan, D C I

    2011-01-01

    The PI3-kinase (PI3K)/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling cascades promote normal growth and are frequently hyperactivated in tumour cells. mTORC1 is also regulated by local nutrients, particularly amino acids, but the mechanisms involved are poorly understood. Unexpectedly, members of the proton-assisted amino acid transporter (PAT or SLC36) family emerged from in vivo genetic screens in Drosophila as transporters with uniquely potent effects on mTORC1-mediated growth. Here we show the two human PATs that are widely expressed in normal tissues and cancer cell lines, PAT1 and PAT4, behave similarly to fly PATs when expressed in Drosophila. siRNA knockdown reveals that these molecules are required for activation of mTORC1 targets and for proliferation in human MCF-7 breast cancer and HEK-293 embryonic kidney cell lines. Furthermore, activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, where mTOR shuttles upon amino acid stimulation. Our data are therefore consistent with a model in which PATs modulate mTORC1's activity not by transporting amino acids into the cell, but by modulating the intracellular response to amino acids. PMID:20498635

  5. Methicillin-resistant Staphylococcus aureus infected gluteal compartment syndrome with rhabdomyolysis in a bodybuilder

    PubMed Central

    Woon, Colin YL; Patel, Kushal R; Goldberg, Benjamin A

    2016-01-01

    Gluteal compartment syndrome (GCS) is a rare condition. We present a case of gluteal muscle strain with hematoma formation, methicillin-resistant Staphylococcus aureus (MRSA) superinfection, leading to acute GCS, rhabdomyolysis and acute kidney injury. This combination of diagnoses has not been reported in the literature. A 36-year-old Caucasian male presented with buttock pain, swelling and fever after lifting weights. Gluteal compartment pressure was markedly elevated compared with the contralateral side. Investigations revealed elevated white blood cell, erythrocyte sedimentation rate, C-reactive protein, creatine kinase, creatinine and lactic acid. Urinalysis was consistent with myoglobinuria. Magnetic resonance imaging showed increased T2 signal in the gluteus maximus and a central hematoma. Cultures taken from the emergency debridement and fasciotomy revealed MRSA. He had repeat, debridement 2 d later, and delayed primary closure 3 d after. GCS is rare and must be suspected when patients present with pain and swelling after an inciting event. They are easily diagnosed with compartment pressure monitoring. The treatment of gluteal abscess and compartment syndrome is the same and involves rapid surgical debridement. PMID:27190761

  6. Methicillin-resistant Staphylococcus aureus infected gluteal compartment syndrome with rhabdomyolysis in a bodybuilder.

    PubMed

    Woon, Colin Yl; Patel, Kushal R; Goldberg, Benjamin A

    2016-05-18

    Gluteal compartment syndrome (GCS) is a rare condition. We present a case of gluteal muscle strain with hematoma formation, methicillin-resistant Staphylococcus aureus (MRSA) superinfection, leading to acute GCS, rhabdomyolysis and acute kidney injury. This combination of diagnoses has not been reported in the literature. A 36-year-old Caucasian male presented with buttock pain, swelling and fever after lifting weights. Gluteal compartment pressure was markedly elevated compared with the contralateral side. Investigations revealed elevated white blood cell, erythrocyte sedimentation rate, C-reactive protein, creatine kinase, creatinine and lactic acid. Urinalysis was consistent with myoglobinuria. Magnetic resonance imaging showed increased T2 signal in the gluteus maximus and a central hematoma. Cultures taken from the emergency debridement and fasciotomy revealed MRSA. He had repeat, debridement 2 d later, and delayed primary closure 3 d after. GCS is rare and must be suspected when patients present with pain and swelling after an inciting event. They are easily diagnosed with compartment pressure monitoring. The treatment of gluteal abscess and compartment syndrome is the same and involves rapid surgical debridement. PMID:27190761

  7. Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments.

    PubMed

    Tanaka, N; Fujita, M; Handa, H; Murayama, S; Uemura, M; Kawamura, Y; Mitsui, T; Mikami, S; Tozawa, Y; Yoshinaga, T; Komatsu, S

    2004-06-01

    Despite recent progress in sequencing the complete genome of rice ( Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60-98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function. PMID:15069638

  8. [Deposition of exogenous and endogenously generated unsaturated fatty acids in lipid droplets triacylglycerol as a mechanism of its sequestration in epithelial cells].

    PubMed

    Fedorova, E V; Fok, E M; Bakhteeva, V T; Lavrova, E A; Parnova, R G

    2014-08-01

    Neutral lipids are deposited in intracellular compartments called lipid droplets, which are known to be critically implicated in regulation of cellular lipid metabolism. These organelles consist of a core of neutral lipids, mainly triacylglycerol (TAG) and cholesteryl esters, surrounded by phospholipid monolayer. Using Nile red lipid staining and [3H]-arachidonic and [3H]-oleic acids as precursors for lipid biosynthesis, we have evaluated the mechanisms of lipid body induction elicited by exogenous fatty acids within primary cultured epithelial cells from the frog urinary bladder. It was found that arachidonic and oleic acids at concentrations 10-50 tM stimulated lipid droplets formation accompanied by accumulation of TAG and by the significant increase of incorporation of fatty acids into TAG indicating an enhanced TAG biosynthesis. No changes of cholesteryl esters content were observed under these conditions. In cells, prelabelled with [3H]-oleic acids, etomoxir, an inhibitor of O-carnitine palmitroyltansferase 1, decreased oxidation of oleic acid and increased its incorporation into TAG leading to intracellular TAG accumulation. In cells, prelabelled with [3H]-arachidonic acid, diclofenac, an inhibitor of cyclooxygenase 1 and 2, led to significant decrease in cellular PGE2 production and to reesterification of free arachidonic acid to TAG but not to phospholipids. Taking together, these data evidence that in isolated frog urinary bladder epithelial cells, reacylation of unsaturated free fatty acids into TAG is a main route of their metabolic conversion under the conditions of the increased cytosolic level of free fatty acids. PMID:25682688

  9. 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

  10. A HaloTag® method for assessing the retrograde axonal transport of the p75 neurotrophin receptor and other proteins in compartmented cultures of rat sympathetic neurons.

    PubMed

    Mok, Sue-Ann; Lund, Karen; Lapointe, Paul; Campenot, Robert B

    2013-03-30

    We have adapted HaloTag® (HT) technology for use in compartmented cultures of rat sympathetic neurons in order to provide a technique that can be broadly applied to studies of the retrograde transport of molecules that play roles in neurotrophin signaling. Transfected neurons expressing HT protein alone, HT protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to tubulin α-1B were maintained in compartmented cultures in which cell bodies and proximal axons of rat sympathetic neurons reside in proximal compartments and their distal axons extend into distal compartments. HT ligand containing a fluorescent tetramethylrhodamine (TMR) label was applied either in the distal compartments or the proximal compartments, and the transport of labeled proteins was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed alone displayed little or no retrograde transport. HT protein fused to either the intracellular C-terminus or the extracellular N-terminus of p75NTR was retrogradely transported. The retrograde transport of p75NTR was augmented when the distal axons were provided with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT protein fused to the N-terminus of tubulin α-1B was also demonstrated. We conclude that retrograde transport of HT fusion proteins provides a powerful and novel approach in studies of axonal transport. PMID:23348044

  11. A general mechanism for intracellular toxicity of metal-containing nanoparticles† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01234h Click here for additional data file.

    PubMed Central

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto

    2014-01-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment – where particles are abundantly internalized – is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a “lysosome-enhanced Trojan horse effect” since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. PMID:24842463

  12. 14 CFR 135.170 - Materials for compartment interiors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Aircraft and Equipment § 135.170 Materials for compartment interiors. (a) No person may operate an airplane... issuance of the initial airworthiness certificate under that SFAR, the airplane meets the compartment... a large airplane unless it meets the following additional airworthiness requirements: (1) Except...

  13. 46 CFR 169.625 - Compartments containing diesel machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery... stresses induced by weight and engine vibration and to minimize transfer of vibration to the...

  14. 14 CFR 25.857 - Cargo compartment classification.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Cargo compartment classification. 25.857 Section 25.857 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Design and Construction Fire Protection § 25.857 Cargo compartment classification....

  15. Compartment Syndrome of the Arm After Cable-Wakeboard Accident.

    PubMed

    Barendse-Hofmann, Minke G; Steenvoorde, Pascal; van Doorn, Louk; Zeillemaker, Anneke

    2009-02-01

    A compartment syndrome is an increased tissue pressure within a closed osteofascial compartment. This compromises blood flow to the muscles and nerves within that compartment, which -if not treated adequately in an early stage-results in permanent tissue and nerve damage. It most frequently occurs in the lower leg, but can also occur elsewhere when muscles are enclosed in tight fascial compartments, such as the forearm and hand. In this report a patient is described who developed an acute compartment syndrome of the arm after a cable-wakeboard accident in which his arm was strangulated. Cable-wakeboarding is an extreme sport that has become very popular over the last years. Early recognition and treatment of an acute compartment syndrome is of extreme importance since in short term necrotic muscles can lead to severe irreversible complications. Accidents with cable-wakeboarding often occur during the start. This is caused by the strong forces that are on the cable during the start. Strangulation injuries of the arm can cause a compartment syndrome of the arm. Possibly a wet-suit or dry-suit offers some protection. However, the duration of strangulation determines much of the damage. Although diagnosis of a compartment syndrome can be difficult, a high index of suspicion combined with fast and adequate treatment with a fasciotomy improve outcome and prognosis. PMID:26814537

  16. 14 CFR 25.857 - Cargo compartment classification.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... access provisions are being used, no hazardous quantity of smoke, flames, or extinguishing agent, will enter any compartment occupied by the crew or passengers; (3) There is a separate approved smoke... compartment but in which— (1) There is a separate approved smoke detector or fire detector system to...

  17. Spontaneous Compartment Syndrome of the Hand in Systemic Sclerosis.

    PubMed

    Tanagho, Andy; Hatab, Sameh; Youssef, Sally; Ansara, Sameh

    2015-09-01

    Compartment syndrome refers to a condition of compromised circulation within a limited space due to increased pressure within that space. The reduced tissue perfusion results in reduced venous drainage, leading to increased interstitial tissue pressure and subsequent compromised arterial flow. Although not as common as compartment syndrome of the leg and forearm, compartment syndrome of the hand is not rare and can lead to devastating sequelae as a result of tissue necrosis. Compartment syndrome of the hand has several etiologies, including trauma, arterial injury, thermal injury, and constrictive bandaging. The cardinal clinical sign is pain that is aggravated by passive stretching of the muscles within the involved compartments. Extremity function is usually restored with expeditious fasciotomy of the involved myofascial compartments, and complications, such as intrinsic muscular dysfunction and Volkmann's ischemic contracture, can usually be prevented. There are no reported cases of compartment syndrome of the hand in patients with systemic sclerosis or Raynaud's phenomenon. Systemic sclerosis is a form of scleroderma that affects the skin and internal organs. The limited cutaneous subset affects the skin of the extremities but is associated with a set of characteristic features that includes calcinosis, Raynaud's phenomenon, esophageal involvement, sclerodactyly, and telangiectasia. This report describes an unusual case of a patient who had spontaneous compartment syndrome of the hand. The patient's concomitant limited cutaneous systemic sclerosis may have played a role in this unusual occurrence. The diagnosis was based on the clinical picture, and the symptoms resolved after surgical decompression. PMID:26375546

  18. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  19. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  20. 46 CFR 169.625 - Compartments containing diesel machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery... stresses induced by weight and engine vibration and to minimize transfer of vibration to the...

  1. 46 CFR 169.625 - Compartments containing diesel machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery... stresses induced by weight and engine vibration and to minimize transfer of vibration to the...

  2. 46 CFR 169.625 - Compartments containing diesel machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery... stresses induced by weight and engine vibration and to minimize transfer of vibration to the...

  3. 14 CFR 29.855 - Cargo and baggage compartments.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Cargo and baggage compartments. 29.855 Section 29.855 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Design and Construction Fire Protection § 29.855 Cargo and baggage compartments. (a)...

  4. Unrecognized acute exertional compartment syndrome of the leg and treatment.

    PubMed

    Popovic, Nebojsa; Bottoni, Craig; Cassidy, Charles

    2011-04-01

    Acute-on-chronic exertional compartment syndrome is rare and may be easily missed without a high degree of awareness and clinical suspicion. We report a case of unrecognized acute-on-chronic exertional compartment syndrome in a recreational soccer player. The late sequela of this condition, foot drop, was successfully treated with transfer of the peroneus longus tendon. PMID:21667742

  5. 14 CFR 25.857 - Cargo compartment classification.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... access provisions are being used, no hazardous quantity of smoke, flames, or extinguishing agent, will enter any compartment occupied by the crew or passengers; (3) There is a separate approved smoke... compartment but in which— (1) There is a separate approved smoke detector or fire detector system to...

  6. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  7. 14 CFR 91.613 - Materials for compartment interiors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Materials for compartment interiors. 91.613 Section 91.613 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... compartment interiors. (a) No person may operate an airplane that conforms to an amended or supplemental...

  8. 46 CFR 169.627 - Compartments containing diesel fuel tanks.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Compartments containing diesel fuel tanks. 169.627 Section 169.627 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Ventilation § 169.627 Compartments containing diesel fuel...

  9. 46 CFR 169.627 - Compartments containing diesel fuel tanks.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Compartments containing diesel fuel tanks. 169.627 Section 169.627 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Ventilation § 169.627 Compartments containing diesel fuel...

  10. 46 CFR 169.627 - Compartments containing diesel fuel tanks.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Compartments containing diesel fuel tanks. 169.627 Section 169.627 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Ventilation § 169.627 Compartments containing diesel fuel...

  11. 46 CFR 169.627 - Compartments containing diesel fuel tanks.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Compartments containing diesel fuel tanks. 169.627 Section 169.627 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Ventilation § 169.627 Compartments containing diesel fuel...

  12. 46 CFR 169.625 - Compartments containing diesel machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Compartments containing diesel machinery. 169.625... SCHOOL VESSELS Machinery and Electrical Ventilation § 169.625 Compartments containing diesel machinery. (a) Spaces containing machinery must be fitted with adequate dripproof ventilators, trunks,...

  13. 14 CFR 25.772 - Pilot compartment doors.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Pilot compartment doors. 25.772 Section 25... § 25.772 Pilot compartment doors. For an airplane that has a lockable door installed between the pilot... passengers require use of the flightdeck door in order to reach the emergency exits provided for them; and...

  14. 14 CFR 25.772 - Pilot compartment doors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Pilot compartment doors. 25.772 Section 25... § 25.772 Pilot compartment doors. For an airplane that has a lockable door installed between the pilot... passengers require use of the flightdeck door in order to reach the emergency exits provided for them; and...

  15. 14 CFR 25.772 - Pilot compartment doors.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Pilot compartment doors. 25.772 Section 25... § 25.772 Pilot compartment doors. For an airplane that has a lockable door installed between the pilot... passengers require use of the flightdeck door in order to reach the emergency exits provided for them; and...

  16. 14 CFR 25.772 - Pilot compartment doors.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Pilot compartment doors. 25.772 Section 25... § 25.772 Pilot compartment doors. For an airplane that has a lockable door installed between the pilot... passengers require use of the flightdeck door in order to reach the emergency exits provided for them; and...

  17. 14 CFR 25.772 - Pilot compartment doors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Pilot compartment doors. 25.772 Section 25... § 25.772 Pilot compartment doors. For an airplane that has a lockable door installed between the pilot... passengers require use of the flightdeck door in order to reach the emergency exits provided for them; and...

  18. 14 CFR 27.787 - Cargo and baggage compartments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... emergency landing conditions of § 27.561. (b) There must be means to prevent the contents of any compartment...) Under the emergency landing conditions of § 27.561, cargo and baggage compartments must— (1) Be... any of the escape facilities provided for use after an emergency landing; or (2) Have...

  19. 14 CFR 29.787 - Cargo and baggage compartments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... emergency landing conditions of § 29.561. (b) There must be means to prevent the contents of any compartment...) Under the emergency landing conditions of § 29.561, cargo and baggage compartments must— (1) Be... any of the escape facilities provided for use after an emergency landing; or (2) Have...

  20. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  1. Cathepsin L Occupies a Vacuolar Compartment and is a Protein Maturase within the Endo/Exocytic System of Toxoplasma gondii

    PubMed Central

    Parussini, Fabiola; Coppens, Isabelle; Shah, Parag P.; Diamond, Scott L.; Carruthers, Vern B.

    2010-01-01

    Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfill their functions. The intracellular parasite Toxoplasma gondii has one of the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized vacuolar compartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite. PMID:20444089

  2. Cell-specific intracellular anticancer drug delivery from mesoporous silica nanoparticles with pH sensitivity.

    PubMed

    Luo, Zhong; Cai, Kaiyong; Hu, Yan; Zhang, Beilu; Xu, Dawei

    2012-05-01

    A nanoreservoir for efficient intracellular anticancer drug delivery based on mesoporous silica nanoparticles end-capped with lactobionic acid-grafted bovine serum albumin is fabricated. It demonstrates great potential for both cell-specific endocytosis and intracellular pH-responsive controlled release of drugs. A possible endocytosis pathway/mechanism of the smart controlled drug release system is proposed. PMID:23184747

  3. Compartment in vertical flow reactor for ferruginous mine water

    NASA Astrophysics Data System (ADS)

    Hur, Won; Cheong, Young-Wook; Yim, Gil-Jae; Ji, Sang-Woo; Hong, Ji-Hye

    2014-05-01

    Mine effluents contain varying concentrations of ferrous ion along with other metal ions. Fe(II) that quickly oxidizes to form precipitates in the presence of oxygen under net alkaline or neutral conditions. Thus, passive treatment methods are designed for the mine water to reside in an open containment area so as to allow simultaneous oxidation and precipitation of Fe(II), such as in a lagoon or an oxidation pond. A vertical flow reactor (VFR) was also suggested to remediate ferruginous mine drainage passing down through an accreting bed of ochre. However, VFR has a limited operation time until the system begins to overflow. It was also demonstrated that two-compartment VFR has a longer operation time than single compartment VFR of same size. In this study, a mathematical model was developed as a part of efforts to explore the operation of VFR, showing dynamic changes in head differences, ochre depth and Fe(II)/Fe(III) concentration in the effluent flow. The analysis shows that Fe(II) oxidation and ochre formation should be balanced with permeability of ochre bed to maximize VFR operation time and minimize residual Fe(II) in the effluent. The model demonstrates that two compartment VFR can have a longer operation time than a single-compartment VFR and that an optimum compartment ratio exists that maximize VFR operation time. Accelerated Fe(II) oxidation significantly affects the optimum ratio of compartment area and reduced residual Fe(II) in the effluent. VFR operation time can be significantly prolonged by increasing the rate of ochre formation not by accelerated Fe(II) oxidation. Taken together, ochre forms largely in the first compartment while overflowed mine water with reduced iron contents is efficiently filtered in the second compartment. These results provide us a better understanding of VFR operation and optimum design criteria for maximum operation time in a two-compartment VFR. Rapid ochre accretion in the first compartment maintains constant hydraulic

  4. 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.

  5. Structural and functional analysis of endosomal compartments in epithelial cells.

    PubMed

    Bay, Andres E Perez; Schreiner, Ryan; Rodriguez-Boulan, Enrique

    2015-01-01

    Epithelial cells display segregated early endosomal compartments, termed apical sorting endosomes and basolateral sorting endosomes, that converge into a common late endosomal-lysosomal degradative compartment and common recycling endosomes (CREs). Unlike recycling endosomes of nonpolarized cells, CREs have the ability to sort apical and basolateral plasma membrane proteins into distinct apical and basolateral recycling routes, utilizing mechanisms similar to those employed by the trans Golgi network in the biosynthetic pathway. The apical recycling route includes an additional compartment, the apical recycling endosomes, consisting of multiple vesicles bundled around the basal body. Recent evidence indicates that, in addition to their role in internalizing ligands and recycling their receptors back to the cell surface, endosomal compartments act as intermediate stations in the biosynthetic routes to the plasma membrane. Here we review methods employed by our laboratory to study the endosomal compartments of epithelial cells and their multiple trafficking roles. PMID:26360040

  6. Safe arthroscopic access to the central compartment of the hip.

    PubMed

    Dienst, Michael; Seil, Romain; Kohn, Dieter M

    2005-12-01

    This technical note describes a new method that allows access to the central compartment of the hip under arthroscopic control via the peripheral compartment with less risk of labral perforation and/or cartilage scuffing. After placement of the anterolateral portal in the peripheral compartment without traction, the anterior head area with the anterior acetabular labrum and the anterior surface of the femoral head are inspected. Under arthroscopic control, a guidewire is introduced through the anterior portal in between the anterior labrum and anterior femoral head cartilage and into the central compartment. The arthroscope is then removed from the anterolateral portal, the hip distracted, and the arthroscope introduced via cannulated instruments over the guidewire into the central compartment. Further portal placement can be controlled arthroscopically. PMID:16376244

  7. Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

    PubMed

    Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

    2007-06-01

    Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown. PMID:17264222

  8. 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.

  9. 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.

  10. A single amino acid difference between the intracellular domains of amyloid precursor protein and amyloid-like precursor protein 2 enables induction of synaptic depression and block of long-term potentiation.

    PubMed

    Trillaud-Doppia, Emilie; Paradis-Isler, Nicolas; Boehm, Jannic

    2016-07-01

    Alzheimer disease (AD) is initially characterized as a disease of the synapse that affects synaptic transmission and synaptic plasticity. While amyloid-beta and tau have been traditionally implicated in causing AD, recent studies suggest that other factors, such as the intracellular domain of the amyloid-precursor protein (APP-ICD), can also play a role in the development of AD. Here, we show that the expression of APP-ICD induces synaptic depression, while the intracellular domain of its homolog amyloid-like precursor protein 2 (APLP2-ICD) does not. We are able to show that this effect by APP-ICD is due to a single alanine vs. proline difference between APP-ICD and APLP2-ICD. The alanine in APP-ICD and the proline in APLP2-ICD lie directly behind a conserved caspase cleavage site. Inhibition of caspase cleavage of APP-ICD prevents the induction of synaptic depression. Finally, we show that the expression of APP-ICD increases and facilitates long-term depression and blocks induction of long-term potentiation. The block in long-term potentiation can be overcome by mutating the aforementioned alanine in APP-ICD to the proline of APLP2. Based on our results, we propose the emergence of a new APP critical domain for the regulation of synaptic plasticity and in consequence for the development of AD. PMID:26921470

  11. 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).

  12. Medial Compartment Decompression by Fibular Osteotomy to Treat Medial Compartment Knee Osteoarthritis: A Pilot Study.

    PubMed

    Yang, Zong-You; Chen, Wei; Li, Cun-Xiang; Wang, Juan; Shao, De-Cheng; Hou, Zhi-Yong; Gao, Shi-Jun; Wang, Fei; Li, Ji-Dong; Hao, Jian-Dong; Chen, Bai-Cheng; Zhang, Ying-Ze

    2015-12-01

    Compared with high tibial osteotomy and total knee arthroplasty, the authors found a simpler surgical procedure, partial fibular osteotomy, could effectively relieve knee pain and also correct the varus deformity for patients with medial compartment knee osteoarthritis (OA). From January 1996 to April 2012, a total of 156 patients with medial compartment OA were treated by proximal fibular osteotomy in the authors' hospital. A 2-cm-long section of fibula was resected 6 to 10 cm below the fibular head. A total of 110 patients with follow-up of more than 2 years were included in the study, including 34 males and 76 females with an average age of 59.2 years. Anteroposterior and lateral weight-bearing radiographs, the femorotibial angle (FTA) and lateral joint space, and the American Knee Society Score (KSS) and the visual analog scale (VAS) score of the knee joint were evaluated preoperatively and at final follow-up, respectively. At final follow-up, mean FTA and lateral joint space were 179.4°±1.8° and 6.9±0.7 mm, respectively, which were significantly smaller than those measured preoperatively (182.7°±2.0° and 12.2±1.1 mm, respectively; both P<.001). Mean KSS at final follow-up was 92.3±31.7, significantly higher than the mean preoperative score of 45.0±21.3 (P<.001). Mean VAS score and interquartile range were 2.0 and 2.0, significantly lower than the preoperative data (7 and 1.0, respectively; P<.001). The authors found that proximal fibular osteotomy can significantly improve both the radiographic appearance and function of the affected knee joint and also achieve long-term pain relief. This procedure may be an alternative treatment option for medial compartment OA. PMID:26652332

  13. An intrinsically disordered domain has a dual function coupled to compartment-dependent redox control.

    PubMed

    Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Ciofi-Baffoni, Simone; Gajda, Karolina; Felli, Isabella C; Gallo, Angelo; Pavelkova, Anna; Kallergi, Emmanouela; Andreadaki, Maria; Katrakili, Nitsa; Pozidis, Charalambos; Tokatlidis, Kostas

    2013-02-01

    The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria. At variance to the unfolded-to-folded structural transition of several intrinsically disordered proteins, neither substrate recognition events nor redox switch of its shuttle cysteine pair is linked to any such structural change. However, this unstructured domain performs a dual function in two cellular compartments: it acts (i) as a mitochondrial targeting signal in the cytosol and (ii) as a crucial recognition site in the disulfide relay system of intermembrane space. This domain provides an exciting new paradigm for IDDs ensuring two distinct functions that are linked to intracellular organelle targeting. PMID:23207295

  14. 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

  15. Intracellular processing of the Newcastle disease virus fusion glycoprotein

    SciTech Connect

    Morrison, T.; Ward, L.J.; Semerjian, A.

    1985-03-01

    The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with (/sup 3/H) fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.

  16. 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

  17. Acute compartment syndrome of the foot in a soccer player: a case report

    PubMed Central

    Laframboise, Michelle A.; Muir, Brad

    2011-01-01

    Objective To present the diagnostic and clinical features including management of acute compartment syndrome (ACS) of the foot and to create a sense of emergency amongst clinicians of this rare and dangerous condition. Clinical Features A 28-year old male soccer player on acetylsalicylic acid (ASA) and verapamil presented with severe swelling, paresthesia, and pain in the left ankle after an acute grade three-inversion ankle sprain. A diagnosis of foot compartment syndrome was made. Intervention and Outcome A fasciotomy was not performed and subsequent neurological sequelae occurred. We hypothesize that the edema caused by the ankle sprain was excessive due to the use of ASA and verapamil, resulting in increased compartmental pressure and neurological signs in the foot. Summary Although rare, it is extremely important to be aware of the clinical features of ACS of the foot to obtain an appropriate diagnosis and manage this medical emergency promptly. PMID:22131567

  18. Hydrogen Peroxide Probes Directed to Different Cellular Compartments

    PubMed Central

    Malinouski, Mikalai; Zhou, You; Belousov, Vsevolod V.; Hatfield, Dolph L.; Gladyshev, Vadim N.

    2011-01-01

    Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. PMID:21283738

  19. 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

  20. Striatal patch compartment lesions reduce stereotypy following repeated cocaine administration.

    PubMed

    Murray, Ryan C; Logan, Mary C; Horner, Kristen A

    2015-08-27

    Stereotypy can be characterized as inflexible, repetitive behaviors that occur following repeated exposure to psychostimulants, such as cocaine (COC). Stereotypy may be related to preferential activation of the patch (striosome) compartment of striatum, as enhanced relative activation of the patch compartment has been shown to positively correlate with the emergence of stereotypy following repeated psychostimulant treatment. However, the specific contribution of the patch compartment to COC-induced stereotypy following repeated exposure is unknown. To elucidate the involvement of the patch compartment to the development of stereotypy following repeated COC exposure, we determined if destruction of this sub-region altered COC-induced behaviors. The neurons of the patch compartment were ablated by bilateral infusion of the neurotoxin dermorphin-saporin (DERM-SAP; 17 ng/μl) into the striatum. Animals were allowed to recover for eight days following the infusion, and then were given daily injections of COC (25mg/kg) or saline for one week, followed by a weeklong drug-free period. Animals were then given a challenge dose of saline or COC, observed for 2h in activity chambers and sacrificed. The number of mu-labeled patches in the striatum were reduced by DERM-SAP pretreatment. In COC-treated animals DERM-SAP pretreatment significantly reduced the immobilization and intensity of stereotypy but increased locomotor activity. DERM-SAP pretreatment attenuated COC-induced c-Fos expression in the patch compartment, while enhancing COC-induced c-Fos expression in the matrix compartment. These data indicate that the patch compartment contributes to repetitive behavior and suggests that alterations in activity in the patch vs matrix compartments may underlie to this phenomenon. PMID:26100338

  1. Compartment-dependent mitochondrial alterations in experimental ALS, the effects of mitophagy and mitochondriogenesis.

    PubMed

    Natale, Gianfranco; Lenzi, Paola; Lazzeri, Gloria; Falleni, Alessandra; Biagioni, Francesca; Ryskalin, Larisa; Fornai, Francesco

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by massive loss of motor neurons. Data from ALS patients and experimental models indicate that mitochondria are severely damaged within dying or spared motor neurons. Nonetheless, recent data indicate that mitochondrial preservation, although preventing motor neuron loss, fails to prolong lifespan. On the other hand, the damage to motor axons plays a pivotal role in determining both lethality and disease course. Thus, in the present article each motor neuron compartment (cell body, central, and peripheral axons) of G93A SOD-1 mice was studied concerning mitochondrial alterations as well as other intracellular structures. We could confirm the occurrence of ALS-related mitochondrial damage encompassing total swelling, matrix dilution and cristae derangement along with non-pathological variations of mitochondrial size and number. However, these alterations occur to a different extent depending on motor neuron compartment. Lithium, a well-known autophagy inducer, prevents most pathological changes. However, the efficacy of lithium varies depending on which motor neuron compartment is considered. Remarkably, some effects of lithium are also evident in wild type mice. Lithium is effective also in vitro, both in cell lines and primary cell cultures from the ventral spinal cord. In these latter cells autophagy inhibition within motor neurons in vitro reproduced ALS pathology which was reversed by lithium. Muscle and glial cells were analyzed as well. Cell pathology was mostly severe within peripheral axons and muscles of ALS mice. Remarkably, when analyzing motor axons of ALS mice a subtotal clogging of axoplasm was described for the first time, which was modified under the effects of lithium. The effects induced by lithium depend on several mechanisms such as direct mitochondrial protection, induction of mitophagy and mitochondriogenesis. In this study, mitochondriogenesis induced by lithium was confirmed in situ by a

  2. Compartment-dependent mitochondrial alterations in experimental ALS, the effects of mitophagy and mitochondriogenesis

    PubMed Central

    Natale, Gianfranco; Lenzi, Paola; Lazzeri, Gloria; Falleni, Alessandra; Biagioni, Francesca; Ryskalin, Larisa; Fornai, Francesco

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by massive loss of motor neurons. Data from ALS patients and experimental models indicate that mitochondria are severely damaged within dying or spared motor neurons. Nonetheless, recent data indicate that mitochondrial preservation, although preventing motor neuron loss, fails to prolong lifespan. On the other hand, the damage to motor axons plays a pivotal role in determining both lethality and disease course. Thus, in the present article each motor neuron compartment (cell body, central, and peripheral axons) of G93A SOD-1 mice was studied concerning mitochondrial alterations as well as other intracellular structures. We could confirm the occurrence of ALS-related mitochondrial damage encompassing total swelling, matrix dilution and cristae derangement along with non-pathological variations of mitochondrial size and number. However, these alterations occur to a different extent depending on motor neuron compartment. Lithium, a well-known autophagy inducer, prevents most pathological changes. However, the efficacy of lithium varies depending on which motor neuron compartment is considered. Remarkably, some effects of lithium are also evident in wild type mice. Lithium is effective also in vitro, both in cell lines and primary cell cultures from the ventral spinal cord. In these latter cells autophagy inhibition within motor neurons in vitro reproduced ALS pathology which was reversed by lithium. Muscle and glial cells were analyzed as well. Cell pathology was mostly severe within peripheral axons and muscles of ALS mice. Remarkably, when analyzing motor axons of ALS mice a subtotal clogging of axoplasm was described for the first time, which was modified under the effects of lithium. The effects induced by lithium depend on several mechanisms such as direct mitochondrial protection, induction of mitophagy and mitochondriogenesis. In this study, mitochondriogenesis induced by lithium was confirmed in situ by a

  3. Blood volume and body fluid compartments in lambs with aortopulmonary left-to-right shunts.

    PubMed Central

    Gratama, J W; Dalinghaus, M; Meuzelaar, J J; Gerding, A M; Koers, J H; Zijlstra, W G; Kuipers, J R

    1992-01-01

    A left-to-right shunt is accompanied by an increased plasma and blood volume. Since this is likely realized through renin/aldosterone-mediated salt and water retention, other body fluid compartments may be changed too. Therefore, we studied blood volume and body fluid compartments by a single-injection, triple-indicator dilution technique in nine 8-wk-old lambs with an aortopulmonary left-to-right shunt (55 +/- 3% of left ventricular output; mean +/- SEM) and in 11 control lambs, 2.5 wk after surgery. Systemic blood flow was maintained at the same level as in control lambs, but the aortic pressure of the shunt lambs was lower. Blood volume in shunt lambs was larger than in control lambs (110 +/- 6 vs. 84 +/- 7 ml/kg, P < 0.001) through an increase in plasma volume, which correlated significantly with the magnitude of the left-to-right shunt (r = 0.81, P < 0.01). Red blood cell volume was equal to that of control lambs. Evidence was obtained that the increase in plasma volume was induced by a transient increase in renin (8.0 +/- 2.2 vs. 1.6 +/- 0.2 nmol.l-1.h-1; P < 0.02) and aldosterone (0.51 +/- 0.14 vs. 0.24 +/- 0.09 nmol/liter) concentrations. Interstitial water volume, however, was not significantly different from that in control lambs. The amount of intravascular protein was significantly higher than in control lambs (5.0 +/- 0.3 vs. 3.5 +/- 0.2 g/kg body mass, P < 0.001). There were no significant differences in intracellular and total body water volumes between the two groups. We conclude that the increased amount of intravascular protein confines the fluid retained by the kidneys to the vascular compartment. PMID:1430202

  4. Metabolic coupling in urothelial bladder cancer compartments and its correlation to tumor aggressiveness.

    PubMed

    Afonso, Julieta; Santos, Lúcio L; Morais, António; Amaro, Teresina; Longatto-Filho, Adhemar; Baltazar, Fátima

    2016-02-01

    Monocarboxylate transporters (MCTs) are vital for intracellular pH homeostasis by extruding lactate from highly glycolytic cells. These molecules are key players of the metabolic reprogramming of cancer cells, and evidence indicates a potential contribution in urothelial bladder cancer (UBC) aggressiveness and chemoresistance. However, the specific role of MCTs in the metabolic compartmentalization within bladder tumors, namely their preponderance on the tumor stroma, remains to be elucidated. Thus, we evaluated the immunoexpression of MCTs in the different compartments of UBC tissue samples (n = 111), assessing the correlations among them and with the clinical and prognostic parameters. A significant decrease in positivity for MCT1 and MCT4 occurred from normoxic toward hypoxic regions. Significant associations were found between the expression of MCT4 in hypoxic tumor cells and in the tumor stroma. MCT1 staining in normoxic tumor areas, and MCT4 staining in hypoxic regions, in the tumor stroma and in the blood vessels were significantly associated with UBC aggressiveness. MCT4 concomitant positivity in hypoxic tumor cells and in the tumor stroma, as well as positivity in each of these regions concomitant with MCT1 positivity in normoxic tumor cells, was significantly associated with an unfavourable clinicopathological profile, and predicted lower overall survival rates among patients receiving platinum-based chemotherapy. Our results point to the existence of a multi-compartment metabolic model in UBC, providing evidence of a metabolic coupling between catabolic stromal and cancer cells' compartments, and the anabolic cancer cells. It is urgent to further explore the involvement of this metabolic coupling in UBC progression and chemoresistance. PMID:26636903

  5. Toward intracellular targeted delivery of cancer therapeutics: progress and clinical outlook for brain tumor therapy.

    PubMed

    Pandya, Hetal; Debinski, Waldemar

    2012-08-01

    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

  6. Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.

    PubMed

    Fonfria, Elena; Donald, Sarah; Cadd, Verity A

    2016-01-01

    Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments. PMID:26329879

  7. Compartment Syndrome of the Calf Due to Nicolau Syndrome

    PubMed Central

    Enshaei, Ali; Afshar, Ahmadreza

    2016-01-01

    We report a case of Nicolau syndrome in a 15 months old girl following an intramuscular injection of penicillin 6.3.3 in her left buttock. This case is unique because she developed compartment syndrome in her left calf far from her injection site. Her toe’s tips gangrened in the course of her ailment. We hypothesized that the compartment syndrome might be produced by a probable intra-arterial injection that had produced embolic obstruction of the small and medium size arteries in her leg or a probable perineural or periarteial injection had produced secondary sympathetic stimulation, extensive vasospasm, compromised microcirculation and the development of compartment syndrome. PMID:26894227

  8. Cellular oxygen sensing: Importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2

    SciTech Connect

    Steinhoff, Amrei; Pientka, Friederike Katharina; Moeckel, Sylvia; Kettelhake, Antje; Hartmann, Enno; Koehler, Matthias; Depping, Reinhard

    2009-10-02

    Hypoxia-inducible factors are crucial in the regulatory process of oxygen homeostasis of vertebrate cells. Inhibition of prolyl hydroxylation of HIF-{alpha} subunits by prolyl-hydroxylases (PHD1, PHD2 and PHD3) leads to transcription of a greater number of hypoxia responsive genes. We have investigated the subcellular distribution and the molecular mechanisms regulating the intracellular allocation of PHD1 and PHD2. As reported earlier we find PHD1 located exclusively in the nucleus. We demonstrate that nuclear import of PHD1 occurs importin {alpha}/{beta} dependently and relies on a nuclear localisation signal (NLS). By contrast PHD2 is cycling between nucleus and cytoplasm, and nuclear import seems to be independent of 'classical' importin {alpha}/{beta} receptors. Furthermore, we reveal that the exit of PHD2 from the nucleus requires CRM1 and the N-terminal 100 amino acids of the protein. Our findings provide new insights into the mechanisms of the regulation of the oxygen sensor cascade of PHDs in different cellular compartments.

  9. Nanomedicines Eradicating Cancer Stem-like Cells in Vivo by pH-Triggered Intracellular Cooperative Action of Loaded Drugs.

    PubMed

    Kinoh, Hiroaki; Miura, Yutaka; Chida, Tsukasa; Liu, Xueying; Mizuno, Kazue; Fukushima, Shigeto; Morodomi, Yosuke; Nishiyama, Nobuhiro; Cabral, Horacio; Kataoka, Kazunori

    2016-06-28

    Nanomedicines capable of control over drug functions have potential for developing resilient therapies, even against tumors harboring recalcitrant cancer stem cells (CSCs). By coordinating drug interactions within the confined inner compartment of core-shell nanomedicines, we conceived multicomponent nanomedicines directed to achieve synchronized and synergistic drug cooperation within tumor cells as a strategy for enhancing efficacy, overcoming drug resistance, and eradicating CSCs. The approach was validated by using polymeric micellar nanomedicines co-incorporating the pan-kinase inhibitor staurosporine (STS), which was identified as the most potent CSC inhibitor from a panel of signaling-pathway inhibitors, and the cytotoxic agent epirubicin (Epi), through rationally contriving the affinity between the drugs. The micelles released both drugs simultaneously, triggered by acidic endosomal pH, attaining concurrent intracellular delivery, with STS working as a companion for Epi, down-regulating efflux transporters and resistance mechanisms induced by Epi. These features prompted the nanomedicines to eradicate orthotopic xenografts of Epi-resistant mesothelioma bearing a CSC subpopulation. PMID:27093466

  10. 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

  11. Work-Related Pain in Extrinsic Finger Extensor Musculature of Instrumentalists Is Associated with Intracellular pH Compartmentation during Exercise

    PubMed Central

    Moreno-Torres, Angel; Rosset-Llobet, Jaume; Pujol, Jesus; Fàbregas, Sílvia; Gonzalez-de-Suso, Jose-Manuel

    2010-01-01

    Background Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy (31P-MRS). We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism. Methodology/Principal Findings We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls). We used 31P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr), inorganic phosphate (Pi), Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in 31P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition. Conclusions/Significance Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by 31P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself. PMID:20161738

  12. 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

  13. Coenzyme Q10 and α-lipoic acid: antioxidant and pro-oxidant effects in plasma and peripheral blood lymphocytes of supplemented subjects

    PubMed Central

    Silvestri, Sonia; Orlando, Patrick; Armeni, Tatiana; Padella, Lucia; Brugè, Francesca; Seddaiu, Giovanna; Littarru, Gian Paolo; Tiano, Luca

    2015-01-01

    Reactive oxygen species not only cause damage but also have a physiological role in the protection against pathogens and in cell signalling. Mitochondrial nutrients, such as coenzyme Q10 and α-lipoic acid, beside their acknowledged antioxidant activities, show interesting features in relation to their redox state and consequent biological activity. In this study, we tested whether oral supplementation with 200 mg/day of coenzyme Q10 alone or in association with 200 mg/die of α-lipoic acid for 15 days on 16 healthy subjects was able to modulate the oxidative status into different compartments (plasma and cells), in basal condition and following an oxidative insult in peripheral blood lymphocytes exposed in vitro to H2O2. Data have shown that tested compounds produced antioxidant and bioenergetic effects improving oxidative status of the lipid compartment and mitochondrial functionality in peripheral blood lymphocytes. Simultaneously, an increased intracellular reactive oxygen species level was observed, although they did not lead to enhanced DNA oxidative damage. Coenzyme Q10 and α-lipoic acid produced beneficial effects also steering intracellular redox poise toward a pro-oxidant environment. In contrast with other antioxidant molecules, pro-oxidant activities of tested mitochondrial nutrients and consequent oxidant mediated signalling, could have important implications in promoting adaptive response to oxidative stress. PMID:26236096

  14. 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.

  15. 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

  16. 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

  17. Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

    PubMed Central

    Rikihisa, Yasuko

    2011-01-01

    Summary: Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease. PMID:21734244

  18. 113. INTERIOR COMMUNICATIONS COMPARTMENT PORT LOOKING TO STARBOARD SHOWING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    113. INTERIOR COMMUNICATIONS COMPARTMENT - PORT LOOKING TO STARBOARD SHOWING MASTER GYRO AND INTERIOR COMMUNICATIONS SWITCHBOARD. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  19. Decompression of Neglected Compartment Syndrome of the Arm

    PubMed Central

    Fletcher, Matt DA

    2015-01-01

    Introduction: Compartment syndrome of the arm is rare, and delay in diagnosis or treatment can be devastating. A case is reported of compartment syndrome of the whole arm occurring in a remote location and arrival at hospital 15 hours after injury with good results despite delayed treatment. Case Report: A 26 year old male presented with a combined crush injury and compartment syndrome of the arm, with delay in presentation due to remote injury and delay in rescue. Late (>16 hours)fasciotomy was performed with retention of the upper arm, elbow and proximal forearm with good residual function. Conclusion: Despite significant delay in treatment of compartment syndrome of the arm, useful function and tissue can be preserved with adequate decompression. PMID:27299052

  20. 10. Interior view of communications compartment. View toward front of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Interior view of communications compartment. View toward front of aircraft. - Offutt Air Force Base, Looking Glass Airborne Command Post, Looking Glass Aircraft, On Operational Apron covering northeast half of Project Looking Glass Historic District, Bellevue, Sarpy County, NE

  1. 11. Interior view of communications compartment. View toward rear of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Interior view of communications compartment. View toward rear of aircraft. - Offutt Air Force Base, Looking Glass Airborne Command Post, Looking Glass Aircraft, On Operational Apron covering northeast half of Project Looking Glass Historic District, Bellevue, Sarpy County, NE

  2. 9. Interior view of electronics compartment. View toward rear of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Interior view of electronics compartment. View toward rear of aircraft. - Offutt Air Force Base, Looking Glass Airborne Command Post, Looking Glass Aircraft, On Operational Apron covering northeast half of Project Looking Glass Historic District, Bellevue, Sarpy County, NE

  3. 14 CFR 23.787 - Baggage and cargo compartments.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... any compartment, located aft of the occupants and separated by structure, when the ultimate forward... resulting from the ultimate static load factors of § 23.561(b)(3), assuming the maximum allowed baggage...

  4. 14 CFR 23.787 - Baggage and cargo compartments.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... any compartment, located aft of the occupants and separated by structure, when the ultimate forward... resulting from the ultimate static load factors of § 23.561(b)(3), assuming the maximum allowed baggage...

  5. 14 CFR 23.787 - Baggage and cargo compartments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... any compartment, located aft of the occupants and separated by structure, when the ultimate forward... resulting from the ultimate static load factors of § 23.561(b)(3), assuming the maximum allowed baggage...

  6. Dynamics of the Establishment of Multinucleate Compartments in Fusarium oxysporum

    PubMed Central

    Shahi, Shermineh; Beerens, Bas; Manders, Erik M. M.

    2014-01-01

    Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment. PMID:25398376

  7. Dynamics of the establishment of multinucleate compartments in Fusarium oxysporum.

    PubMed

    Shahi, Shermineh; Beerens, Bas; Manders, Erik M M; Rep, Martijn

    2015-01-01

    Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment. PMID:25398376

  8. 3. INCLINE PLANE CAR INTERIOR, UPPER COMPARTMENT. Monongahela Incline ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. INCLINE PLANE CAR INTERIOR, UPPER COMPARTMENT. - Monongahela Incline Plane, Connecting North side of Grandview Avenue at Wyoming Street with West Carson Street near Smithfield Street, Pittsburgh, Allegheny County, PA

  9. Astronaut Richard Covey working in the Crew Compartment Trainer

    NASA Technical Reports Server (NTRS)

    1986-01-01

    Astronaut Richard O. Covey retrieves a helmet from a stowed extravehicular mobility unit (EM) spacesuit in the airlock of the one-G crew compartment trainer (CCT) at JSC. Covey was training for the STS 26 flight.

  10. [Compartment syndrome following laparoscopic procedures in the lithotomy position].

    PubMed

    Donckers, Janneke; de Kruif, Jan H; van der Stappen, William A H

    2010-01-01

    A laparoscopic tubectomy in the lithotomy position was performed on a healthy 31-year-old woman, as treatment following an extra-uterine pregnancy. The operation proceeded without complications and took 60 minutes. However, on the third day following surgery the woman was diagnosed with compartment syndrome, which was treated with three-compartment fasciotomy. Compartment syndrome is a rare but dangerous complication of an operation in the lithotomy position. Since pressure on leg compartments increases with time spent in the lithotomy position, regardless of the type of stirrups used, it is important to maintain the position only as long as is necessary for the procedure. The patient's legs should be taken out of the lithotomy position as soon as possible, and the position resumed if necessary at a later stage in the procedure. This can easily be achieved with pneumatic stirrups. PMID:20619013

  11. Two clinical tests for assessing lateral compartment arthritis.

    PubMed

    Shakespeare, David

    2006-08-01

    Two clinical tests, the valgus tap test and the valgus skid test, are described which detect bone contact in the lateral compartment of the knee. They are useful in planning surgical intervention. PMID:16632364

  12. Diagnosis and treatment of acute extremity compartment syndrome.

    PubMed

    von Keudell, Arvind G; Weaver, Michael J; Appleton, Paul T; Appelton, Paul T; Bae, Donald S; Dyer, George S M; Heng, Marilyn; Jupiter, Jesse B; Vrahas, Mark S

    2015-09-26

    Acute compartment syndrome of the extremities is well known, but diagnosis can be challenging. Ineffective treatment can have devastating consequences, such as permanent dysaesthesia, ischaemic contractures, muscle dysfunction, loss of limb, and even loss of life. Despite many studies, there is no consensus about the way in which acute extremity compartment syndromes should be diagnosed. Many surgeons suggest continuous monitoring of intracompartmental pressure for all patients who have high-risk extremity injuries, whereas others suggest aggressive surgical intervention if acute compartment syndrome is even suspected. Although surgical fasciotomy might reduce intracompartmental pressure, this procedure also carries the risk of long-term complications. In this paper in The Lancet Series about emergency surgery we summarise the available data on acute extremity compartment syndrome of the upper and lower extremities in adults and children, discuss the underlying pathophysiology, and propose a clinical guideline based on the available data. PMID:26460664

  13. 2. INTERIOR, SOUTHWEST VIEW (STORAGE COMPARTMENTS). Vanadium Corporation of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. INTERIOR, SOUTHWEST VIEW (STORAGE COMPARTMENTS). - Vanadium Corporation of America (VCA) Naturita Mill, Mine Warehouse, 3 miles Northwest of Naturita, between Highway 141 & San Miguel River, Naturita, Montrose County, CO

  14. Turbofan Engine Core Compartment Vent Aerodynamic Configuration Development Methodology

    NASA Technical Reports Server (NTRS)

    Hebert, Leonard J.

    2006-01-01

    This paper presents an overview of the design methodology used in the development of the aerodynamic configuration of the nacelle core compartment vent for a typical Boeing commercial airplane together with design challenges for future design efforts. Core compartment vents exhaust engine subsystem flows from the space contained between the engine case and the nacelle of an airplane propulsion system. These subsystem flows typically consist of precooler, oil cooler, turbine case cooling, compartment cooling and nacelle leakage air. The design of core compartment vents is challenging due to stringent design requirements, mass flow sensitivity of the system to small changes in vent exit pressure ratio, and the need to maximize overall exhaust system performance at cruise conditions.

  15. FEATURE 3, LARGE GUN POSITION, SHOWING MULTIPLE COMPARTMENTS, VIEW FACING ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    FEATURE 3, LARGE GUN POSITION, SHOWING MULTIPLE COMPARTMENTS, VIEW FACING SOUTH. - Naval Air Station Barbers Point, Anti-Aircraft Battery Complex-Large Gun Position, East of Coral Sea Road, northwest of Hamilton Road, Ewa, Honolulu County, HI

  16. 19 CFR 123.24 - Sealing of conveyances or compartments.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...; DEPARTMENT OF THE TREASURY CUSTOMS RELATIONS WITH CANADA AND MEXICO Shipments in Transit Through Canada or Mexico § 123.24 Sealing of conveyances or compartments. (a) Sealing required. Merchandise in...

  17. Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

    SciTech Connect

    Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

    1987-05-01

    We studied total cell-associated A14-(/sup 125/I)insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients (2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-(/sup 125/I) insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-(/sup 125/I)insulin, (/sup 125/I)iodide, or (/sup 125/I)tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients.

  18. Abdominal Compartment Syndrome: Risk Factors, Diagnosis, and Current Therapy

    PubMed Central

    Luckianow, Gina M.; Ellis, Matthew; Governale, Deborah; Kaplan, Lewis J.

    2012-01-01

    Abdominal compartment syndrome's manifestations are difficult to definitively detect on physical examination alone. Therefore, objective criteria have been articulated that aid the bedside clinician in detecting intra-abdominal hypertension as well as the abdominal compartment syndrome to initiate prompt and potentially life-saving intervention. At-risk patient populations should be routinely monitored and tiered interventions should be undertaken as a team approach to management. PMID:22720147

  19. Acute pediatric leg compartment syndrome in chronic myeloid leukemia.

    PubMed

    Cohen, Eric; Truntzer, Jeremy; Trunzter, Jeremy; Klinge, Steve; Schwartz, Kevin; Schiller, Jonathan

    2014-11-01

    Acute compartment syndrome is an orthopedic surgical emergency and may result in devastating complications in the setting of delayed or missed diagnosis. Compartment syndrome has a variety of causes, including posttraumatic or postoperative swelling, external compression, burns, bleeding disorders, and ischemia-reperfusion injury. Rare cases of pediatric acute compartment syndrome in the setting of acute myeloid leukemia and, even less commonly, chronic myeloid leukemia have been reported. The authors report the first known case of pediatric acute compartment syndrome in a patient without a previously known diagnosis of chronic myeloid leukemia. On initial examination, an 11-year-old boy presented with a 2-week history of progressive left calf pain and swelling after playing soccer. Magnetic resonance imaging scan showed a hematoma in the left superficial posterior compartment. The patient had unrelenting pain, intermittent lateral foot parethesias, and inability to bear weight. Subsequently, he was diagnosed with acute compartment syndrome and underwent fasciotomy and evacuation of a hematoma. Laboratory results showed an abnormal white blood cell count of 440×10(9)/L (normal, 4.4-11×10(9)) and international normalized ratio of 1.3 (normal, 0.8-1.2). Further testing included the BCR-ABL1 fusion gene located on the Philadelphia chromosome, leading to a diagnosis of chronic myeloid leukemia. Monotherapy with imatinib mesylate (Gleevec) was initiated. This report adds another unique case to the growing literature on compartment syndrome in the pediatric population and reinforces the need to consider compartment syndrome, even in unlikely clinical scenarios. PMID:25361367

  20. Differential gene expression in anatomical compartments of the human eye

    PubMed Central

    Diehn, Jennifer J; Diehn, Maximilian; Marmor, Michael F; Brown, Patrick O

    2005-01-01

    Background The human eye is composed of multiple compartments, diverse in form, function, and embryologic origin, that work in concert to provide us with our sense of sight. We set out to systematically characterize the global gene expression patterns that specify the distinctive characteristics of the various eye compartments. Results We used DNA microarrays representing approximately 30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. The distinctive patterns of expression in each compartment could be interpreted in relation to the physiology and cellular composition of each tissue. Notably, the sets of genes selectively expressed in the retina and in the lens were particularly large and diverse. Genes with roles in immune defense, particularly complement components, were expressed at especially high levels in the anterior segment tissues. We also found consistent differences between the gene expression patterns of the macula and peripheral retina, paralleling the differences in cell layer densities between these regions. Based on the hypothesis that genes responsible for diseases that affect a particular eye compartment are likely to be selectively expressed in that compartment, we compared our gene expression signatures with genetic mapping studies to identify candidate genes for diseases affecting the cornea, lens, and retina. Conclusion Through genome-scale gene expression profiling, we were able to discover distinct gene expression 'signatures' for each eye compartment and identified candidate disease genes that can serve as a reference database for investigating the physiology and pathophysiology of the eye. PMID:16168081

  1. Return to activity following fasciotomy for chronic exertional compartment syndrome.

    PubMed

    Irion, Val; Magnussen, Robert A; Miller, Timothy L; Kaeding, Christopher C

    2014-10-01

    Diagnosis of chronic exertional compartment syndrome (CECS) is relatively rare but has been well documented in athletes. There are, however, few reports regarding return to athletic activity after surgery among elite-level athletes. We hypothesized that a majority of elite-level athletes would successfully return to their previous level of competition following fasciotomy for CECS. A retrospective chart review was performed to identify elite-level athletes (collegiate or professional sport participation) who underwent fasciotomy for CECS over a 3-year period. Data collected included sport or activity, treatment and surgical details, time away from sport/activity after surgery, and ability to return to prior level of activity. Six males and seven females were included in the analysis. Patient age ranged from 17 to 24 years with a mean of 19.7 years. Six patients underwent unilateral lower extremity compartment release, and seven underwent bilateral lower extremity compartment release. The anterior and lateral compartments alone were released in 11 patients (84.6%). Two patients (15.4%) underwent four-compartment releases. Eleven patients (84.6%) were able to return to their previous elite level of sport participation at a mean of 10.6 weeks following surgical fasciotomy. Patients who had four-compartment release had a more than 3.5 week average longer return to full sporting activities (p = 0.011). Fasciotomy is effective in allowing elite athletes with CECS to return to sport. PMID:24664450

  2. Compartment Syndrome of the Gluteus Medius Occurred without Bleeding or Trauma: A Case Report

    PubMed Central

    Kong, Gyu-Min; Park, Jun-Ho

    2015-01-01

    Compartment syndrome is an ischemic change resulting from an increase in compartment pressure. Initially, patients present with direct tenderness and swelling, and the weak circulation secondary to compartment syndrome can eventually lead to motor and sensory impairment. If the increase in pressure results in neurological impairment, emergency intervention is required to decompress the compartment. Typically, compartment syndrome develops on forearms or lower legs. The gluteal compartment is rarely the location of compartment syndrome and only a few cases have been presented in the literature with trauma or hematoma. We have treated a patient with gluteal compartment syndrome who presented with no history of trauma or hemorrhage and present that case report here.

  3. MELiSSA third compartment: Nitrosomonas europaea and Nitrobacter winogradskyi axenic cultures in bioreactors

    NASA Astrophysics Data System (ADS)

    Cruvellier, Nelly; Lasseur, Christophe; Poughon, Laurent; Creuly, Catherine; Dussap, Gilles

    Nitrogen is a key element for the life and its balance on Earth is regulated by the nitrogen cycle. This loop includes several steps among which nitrification that permits the transformation of the ammonium into nitrate. The MELiSSA loop is an artificial ecosystem designed for life support systems (LSS). It is based on the carbon and nitrogen cycles and the recycling of the non-edible part of the higher plants and the waste produced by the crew. In this order, all the wastes are collected in the first compartment to degrade them into organic acids and CO2. These compounds are joining the second compartment which is a photoheterotrophic compartment where at the outlet an organic-free medium containing ammonium is produced. This solution will be the substrate of the third compartment where nitrification is done. This compartment has to oxidize the ammonium into nitrate, and this biological reaction needs two steps. In the MELiSSA loop, the nitrification is carried out by two bacteria: Nitrosomonas europaea ATCC® 19718™ which is oxidizing ammonia into nitrite and Nitrobacter winogradskyi ATCC® 25391™ which is producing nitrate from nitrite in the third compartment. These two bacteria are growing in axenic conditions on a fixed bed bioreactor filled with Biostyr® beads. The nitrogen compounds are controlled by Ionic Chromatography and colorimetric titration for each sample. The work presented here deals with the culture of both bacteria in pure cultures and mixed cultures in stirred and aerated bioreactors of different volumes. The first aim of our work is the characterization of the bacteria growth in bioreactors and in the nitrifying fixed-bed column. The experimental results confirm that the growth is slow; the maximal growth rate in suspended cultures is 0.054h-1 for Nitrosomonas europaea and 0.022h-1 for Nitrobacter winogradskyi. Mixed cultures are difficult to control and operate but one could be done for more than 500 hours. The characterization of the

  4. 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

  5. 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

  6. 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

  7. Comparative efficacy of chloramphenicol loaded chondroitin sulfate and dextran sulfate nanoparticles to treat intracellular Salmonella infections.

    PubMed

    Kiruthika, V; Maya, S; Suresh, Maneesha K; Kumar, V Anil; Jayakumar, R; Biswas, Raja

    2015-03-01

    Salmonella Paratyphi A is a food-borne Gram-negative pathogen and a major public health challenge in the developing world. Upon reaching the intestine, S. Paratyphi A penetrates the intestinal epithelial barrier; and infects phagocytes such as macrophages and dendritic cells. S. Paratyphi A surviving within macrophages is protected from the lethal action of antibiotics due to their poor penetration into the intracellular compartments. Hence we have developed chloramphenicol loaded chondroitin sulfate (CS-Cm Nps) and dextran sulfate (DS-Cm Nps) nanoparticles through ionotropic-gelation method for the intracellular delivery of chloramphenicol. The size of these nanoparticles ranged between 100 and 200 nm in diameter. The encapsulation efficiency of both the nanoparticles was found to be around 65%. Both the nanoparticles are found to be non-hemolytic and non-toxic to fibroblast and epithelial cells. The prepared nanoparticles exhibited sustained release of the drug of up to 40% at pH 5 and 20-25% at pH 7.0 after 168 h. The anti-microbial activities of both nanoparticles were tested under in vitro and ex vivo conditions. The delivery of DS-Cm Nps into the intracellular compartments of the macrophages was 4 fold more compared to the CS-Cm Nps which lead to the enhanced intracellular antimicrobial activity of Ds-Cm Nps. Enhanced anti-microbial activity of Ds-Cm Nps was further confirmed in an ex vivo chicken intestine infection model. Our results showed that Cm loaded DS Nps can be used to treat intracellular Salmonella infections. PMID:25645750

  8. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  9. Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

    PubMed Central

    Antoine, J C; Prina, E; Jouanne, C; Bongrand, P

    1990-01-01

    Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment. Images PMID:1689700

  10. 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

  11. 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

  12. Intracellular Phosphate Dynamics in Muscle Measured by Magnetic Resonance Spectroscopy during Hemodialysis.

    PubMed

    Lemoine, Sandrine; Fournier, Thomas; Kocevar, Gabriel; Belloi, Amélie; Normand, Gabrielle; Ibarrola, Danielle; Sappey-Marinier, Dominique; Juillard, Laurent

    2016-07-01

    Of the 600-700 mg inorganic phosphate (Pi) removed during a 4-hour hemodialysis session, a maximum of 10% may be extracted from the extracellular space. The origin of the other 90% of removed phosphate is unknown. This study tested the hypothesis that the main source of phosphate removed during hemodialysis is the intracellular compartment. Six binephrectomized pigs each underwent one 3-hour hemodialysis session, during which the extracorporeal circulation blood flow was maintained between 100 and 150 ml/min. To determine in vivo phosphate metabolism, we performed phosphorous ((31)P) magnetic resonance spectroscopy using a 1.5-Tesla system and a surface coil placed over the gluteal muscle region. (31)P magnetic resonance spectra (repetition time =10 s; echo time =0.35 ms) were acquired every 160 seconds before, during, and after dialysis. During the dialysis sessions, plasma phosphate concentrations decreased rapidly (-30.4 %; P=0.003) and then, plateaued before increasing approximately 30 minutes before the end of the sessions; 16 mmol phosphate was removed in each session. When extracellular phosphate levels plateaued, intracellular Pi content increased significantly (11%; P<0.001). Moreover, βATP decreased significantly (P<0.001); however, calcium levels remained balanced. Results of this study show that intracellular Pi is the source of Pi removed during dialysis. The intracellular Pi increase may reflect cellular stress induced by hemodialysis and/or strong intracellular phosphate regulation. PMID:26561642

  13. 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

  14. 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

  15. 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

  16. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  17. 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

  18. Isolated Chronic Exertional Compartment Syndrome of the Lateral Lower Leg

    PubMed Central

    van Zantvoort, Aniek P.M.; de Bruijn, Johan A.; Winkes, Michiel B.; Dielemans, Jeanne P.; van der Cruijsen-Raaijmakers, Marike; Hoogeveen, Adwin R.; Scheltinga, Marc R.

    2015-01-01

    Background: Exercise-induced lower leg pain may be caused by chronic exertional compartment syndrome (CECS). The anterior (ant-CECS) or deep posterior compartment (dp-CECS) is usually affected. Knowledge regarding CECS of the lateral compartment (lat-CECS) is limited. Purpose: To describe demographic characteristics and symptoms in a consecutive series of patients with isolated CECS of the lateral compartment of the leg. Study Design: Case series; Level of evidence, 4. Methods: Since 2001, patients undergoing dynamic intracompartmental pressure (ICP) measurements for suspected CECS in a single institution were prospectively monitored. Individuals with a history possibly associated with lat-CECS and elevated ICP measurements (Pedowitz criteria) were identified. Exclusion criteria were concomitant ipsilateral ant-CECS/dp-CECS, acute compartment syndrome, recent significant trauma, peroneal nerve entrapment, or vascular claudication. Results: During an 11-year time period, a total of 26 patients with isolated lat-CECS fulfilled study criteria (15 females; median age, 21 years; range, 14-48 years). Frequently identified provocative sports were running (n = 4), walking (n = 4), field hockey (n = 3), soccer (n = 3), and volleyball (n = 2). Exercise-induced lateral lower leg pain (92%) and tightness (42%) were often reported. The syndrome was bilateral in almost two-thirds (62%, n = 16). Delay in diagnosis averaged 24 months (range, 2 months to 10 years). Conclusion: Young patients with exercise-induced pain in the lateral portions of the lower leg may suffer from isolated CECS of the lateral compartment. ICP measurements in the lateral compartment in these patients are recommended. PMID:26740955

  19. Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

    PubMed

    Hertzén, Erika; Johansson, Linda; Kansal, Rita; Hecht, Alexander; Dahesh, Samira; Janos, Marton; Nizet, Victor; Kotb, Malak; Norrby-Teglund, Anna

    2012-01-01

    Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems. PMID:22511985

  20. Intracellular Streptococcus pyogenes in Human Macrophages Display an Altered Gene Expression Profile

    PubMed Central

    Hertzén, Erika; Johansson, Linda; Kansal, Rita; Hecht, Alexander; Dahesh, Samira; Janos, Marton; Nizet, Victor; Kotb, Malak; Norrby-Teglund, Anna

    2012-01-01

    Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems. PMID:22511985