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Molecular Mechanisms of Endocytosis: Trafficking and Functional Requirements for the Transferrin Receptor, Small Interfering RNAs and Dopamine Transporter: A DissertationNavaroli, Deanna M. 30 April 2012 (has links)
Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping.
The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing.
Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.
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Avicin is a potent sphingomyelinase inhibitor that blocks K-Ras plasma membrane interaction and its oncogenic activityGarrido, Christian M. January 2018 (has links)
No description available.
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The role of PI4KB in cellular localization of small GTPasesSadrpour, Parisa 30 August 2022 (has links)
No description available.
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The Effect of K562-IL21-2 Plasma Membrane Particles on the Proliferation of Natural Killer Cells to Fight CancerProphete, Michelle 01 January 2017 (has links) (PDF)
Immunotherapy has emerged as a current and future paradigm of cancer treatment, which utilizes the body’s immune system to eradicate cancer. Natural Killer (NK) cells as part of the innate immune system have immense potential in their anti-tumor cytotoxic activities and host cell surveillance properties. NK cells comprise approximately five to fifteen percent of peripheral blood lymphocytes and can be proliferated in vitro using recently developed methods with co-cultures with feeder cells (derived from engineered tumor cells) or plasma membrane (PM) particles, produced from the fore mentioned feeder cells, in combination with soluble cytokines. For efficient growth and maintenance of these NK cells, Interleukin-2 (IL-2) is utilized. IL-2 in solution, through receptor mediated signaling, stimulates proliferation of T-cells and NK cells. NK cells have lower responsiveness to IL-2 and consequently require a larger systemic dose to stimulate them as opposed to competing cell populations that have higher expression of receptors for IL-2, such as T-cells, which can have the effect of lower effective stimulation of NK cell growth. Such difference in the stimulatory capability of IL-2 toward NK cells and the short circulation lifetime of soluble IL-2 require higher dosages of soluble IL-2 for effective in vivo NK cell proliferation for therapeutic application against cancer, but is toxic. Therefore establishing another form of IL-2 delivery that improves its specific targeting to NK cells would be beneficial and may be crucial for novel therapeutic improvement. The Copik Laboratory has made an IL-2 fusion protein construct having a membrane anchor for expression of membrane-bound IL-2 on K562-41bbl-21 cells (K562-IL21). K562-IL21 cells are selectively recognized by NK cells and stimulate their proliferation and cytotoxicity. Hence, a K562-IL21 membrane–bound IL-2 form should be targeted to NK cells with IL-2 delivery. K562-IL21-2 cells were then used to prepare PM21-2 particles which have the potential to provide NK cell targeted, long-lived form of IL-2 for use as an injectable drug for in vivo adjuvant stimulation of NK cells. The presence of IL-2 on the in the PM21-2 particle product was verified by Western blot, and ELISA. Particle preparations from the modified K562 cells should possess
characteristics that allow them to possibly replace soluble IL-2 and more specifically increase the numbers or anti-tumor activity of NK cell populations. The effect of PM21-2 particles was studied in in vitro culture based experiments, which tested the effectiveness the PM21-2 particles to induce selective NK cells expansion as compared to PM21 particles in the presence or absence of soluble IL-2.
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Untersuchungen zu Nitrat-induzierbaren Proteinen der Plasmamembran von Chlorella saccharophila (Krüger) Nadson / Investigations on nitrate-inducible proteins in the plasma membrane of Chlorella saccharophila (Krueger) NadsonBrechlin, Peter 30 October 2002 (has links)
No description available.
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Der Syntaxin 1-Cluster - Organisation und Dynamik einer supramolekularen Struktur der Plasmamembran / The Syntaxin 1 Cluster - Organisation and Dynamics of a plasmalemmal supramolecular structureSieber, Jochen Josef 04 May 2007 (has links)
No description available.
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Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screeningVicente Carrillo, Alejandro January 2016 (has links)
Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
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Pathogen entry mechanisms and endocytic responses to plasma membrane damageNygård Skalman, Lars January 2017 (has links)
Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair. During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways. Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension. In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair.
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Vliv ethinylestradiolu na Na+, K+ - ATPázu / The effect of ethinylestradiol on Na+, K+ - ATPaseKettnerová, Karolína January 2014 (has links)
This diploma thesis is oriented to analysis of physiological effect of synthetic estrogen ethinylestradiol (EE), which represents the main component of steroid-based substance used in hormonal contraception. From wide range of physiologically important protein molecules, which might be effected by this steroid, thesis focuses to the study of the sodium plus potassium activated, magnesium dependent adenosinetriphosphatase (Na+, K+ - ATPase), which is selectively inhibited by cardiac glycosides such as ouabain (g strophantine). Na+, K+ - ATPase represents an important plasma membrane bound enzyme, which catalyzes the active transport of sodium and potassium across plasma membrane. In the first part of this work, Na+, K+ - ATPase was determined by binding of radioactively labeled selective inhibitor of this enzyme [3H]ouabain, used for this purpose. In the second part of this work, plasma membrane fluidity was analyzed by steady-state fluorescence anisotropy of DPH. The effect of EE on [3H]ouabain binding was studied first under in vitro conditions by using human embryonic kidney cells (HEK293) which were cultivated for 24 hours in the presence of EE in tissue culture medium. Second, the effect of EE was also studied under in vivo conditions, by subcutaneous application of EE to the female rats of Wistar...
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Lipid transport by ABC proteinsPohl, Antje Heide 19 July 2002 (has links)
In eukaryotischen Zellen sind die Lipidspezies häufig asymmetrisch zwischen den Hälften der Plasmamembran verteilt. Insbesondere Phosphatidylserin (PS) weist oft eine ausgeprägte transversale Asymmetrie auf, da es fast ausschliesslich auf die innere Hälfte der Plasmamembran beschränkt ist. In den letzten Jahren wurden mehrere Proteine diskutiert, die Lipide zwischen den Membranhälften transportieren und möglicherweise die transversale Lipidasymmetrie sowie damit verbundene Zelleigenschaften beeinflussen. Im Mittelpunkt der vorliegenden Promotion steht der Auswärtstransport fluoreszierender (C6-NBD-) Lipid-Analoga und endogener Lipide durch das Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), das der ATP Binding Cassette (ABC) Transporter Superfamilie angehört. Interessanter Weise wird für MDR1 Pgp eine ungewöhnlich breite Substratspezifität angenommen. Das anionische Lipid PS war hier von besonderem Interesse, obgleich es in vorhergehenden Arbeiten nicht als MDR1 Pgp Substrat betrachtet wurde. Der Auswärtstransport von Phosphatidylcholin-, Phosphatidylethanolamin-, Glucosylceramid- und Sphingomyelin-Analoga durch MDR1 Pgp konnte in einer humanen Magenkarzinomlinie (EPG85-257), die MDR1 überexprimiert, mittels Fluoreszenzspektroskopie bestätigt werden. Zudem legt die verringerte Akkumulation von Diacylglycerol- und Ceramid-Analoga den Transport dieser Lipidspezies durch MDR1 Pgp nahe. Im Anschluß an die intrazelluläre Markierung mit C6-NBD-PS mittels eines neuen Verfahrens konnte der signifikant erhöhte Auswärtstransport dieses Analogons in MDR1 überexprimierenden Zellen durch Verwendung spezifischer Inhibitoren MDR1 Pgp zugeschrieben werden. In flusscytometrischen Versuchen war die Exponierung von endogenem PS auf der äusseren Membranhälfte von MDR1 überexprimierenden Zellen signifikant höher als in Kontrollzellen. Verringerung der PS-Exponierung durch einen Inhibitor von MDR1 Pgp deutet auf den Transport von endogenem PS durch MDR1 Pgp hin. Zusätzlich wurde hier der Transport von C6-NBD-PS in vier weiteren Zellinien mit verschiedener Spezies- und Gewebezugehörigkeit charakterisiert, die unterschiedliche Mengen an MDR1 Pgp synthetisieren. Wie Experimente in einer BCRP überexprimierenden EPG85-257-Sublinie nahelegen, ist ausser MDR1 Pgp möglicherweise ebenfalls der ABC Halb-Transporter Breast Cancer Resistance Protein (BCRP) am Transport von C6-NBD-PS und an der verstärkten Exponierung von endogenem PS beteiligt. / In eukaryotic cells, the lipid species are frequently distributed asymmetrically between the plasma membrane leaflets. Phosphatidylserine (PS), in particular, often exhibits a distinct transverse asymmetry, being restricted almost exclusively to the inner leaflet. In the past years, several proteins were suggested to transport lipids between the leaflets of a membrane, and to potentially influence transverse lipid asymmetry and related cell properties. This thesis focuses on outward transport of fluorescent (C6-NBD-) lipid analogs and endogenous lipids by the Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), a member of the ATP binding cassette (ABC) transporter superfamily. Interestingly, MDR1 Pgp has been suggested to exhibit an unusually broad substrate specificity. Here, the anionic PS was of particular concern, although previously reported not to be an MDR1 Pgp substrate. In a human gastric carcinoma cell line (EPG85-257) overexpressing MDR1, outward transport of phosphatidylcholine, phosphatidylethanolamine, glucosylceramide and sphingomyelin analogs via MDR1 Pgp was confirmed using fluorescence spectroscopy. In addition, decreased accumulation of analogs of diacylglycerol and ceramide suggest MDR1 Pgp mediated transport of these lipid species. Upon intracellular labelling with C6-NBD-PS using a novel approach, significantly increased outward transport of this analog in MDR1 overexpressing cells could be attributed to MDR1 Pgp by employing specific inhibitors. In a flow cytometry setup, the exposure of endogenous PS on the outer plasma membrane leaflet was significantly elevated in MDR1 overexpressing cells compared to controls. Reduction of PS exposure by an MDR1 Pgp inhibitor suggests transport of endogenous PS by MDR1 Pgp. Transport of C6-NBD-PS was furthermore characterized here in four additional cell lines of different species and tissue origin with varying synthesis levels of MDR1 Pgp. Besides MDR1 Pgp, the ABC half-size transporter Breast Cancer Resistance Protein (BCRP) is possibly also involved in transport of C6-NBD-PS and in increased exposure of endogenous PS, as found in a BCRP overexpressing EPG85-257 subline.
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