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1	Sources of Diradylglycerols Generated During Cell Growth and Phorbol Ester Stimulation in Madin-Darby Canine Kidney Cells Robinson, Mitchell, Warne, Thomas R. 02 August 1991 (has links) The molecular species of diacylglycerol and alkylacylglycerol of Madin-Darby canine Kidney (MDCK) cells were analyzed to determine the sources of diradylglycerols generated during cell growth and phorbol ester stimulation. MDCK cells in log phase growth contained higher levels of diacylglycerol and alkylacylglycerol than confluent cells. Both subclasses of diradylglycerol showed higher levels of saturated and monoenoic species during log phase. Glycerol incorporation into diradylglycerols was increased during growth, consistent with an increase in their synthesis de novo. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, caused an increase in the level of diacylglycerol but not alkylacylglycerol. Log phase MDCK cells showed a greater response to TPA treatment than confluent cells. The molecular species of diacylglycerol generated during stimulation with either TPA or dioctanoylglycerol closely resembled the species of phosphatidylcholine. These results indicate that TPA and synthetic diacylglycerol stimulate endogenous diacylglycerol production through the hydrolysis of phosphatidylcholine. In contrast, the higher content of diacylglycerol and alkylacylglycerol in replicating MDCK cells is the result of an increase in their synthesis de novo. diacylglycerol molecular species phorbol myristate acetate phosphatidylcholine turnover
2	Inhibition of Phorbol Ester-Stimulated Arachidonic Acid Release by Alkylglycerols Robinson, Mitchell, Burdine, Robin, Warne, Thomas R. 09 February 1995 (has links) Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C. alkylglycerol arachidonic acid madin-darby canine kidney monoglyceride phorbol myristate acetate protein kinase C
3	Establishment of a Long Term Cell Culture Model for Testing Anti-Infectives against Mycobacterium avium subsp. paratuberculosis Kimsawatde, Gade Carolyn 05 May 2015 (has links) Mycobacterium avium subsp. paratuberculosis (MAP) is a very slow growing bacterium that is the causative agent of Johne's disease (JD) in ruminants and has long been suggested to be associated with complications of Crohn's disease (CD) in humans. Although there is no direct evidence that MAP is the primary etiological agent for CD, most CD patients are found to have MAP in their intestinal tissues. The current control measures for JD in cattle, sheep, and goats have only been minimally effective, and there are only medications to treat the symptoms of mycobacterial infections associated with CD in humans. Along with not being able to cure MAP infections, there is no established laboratory animal model for testing therapeutics. When mice are infected with MAP they develop systemic infection and do not mimic disease observed in ruminants. J774A.1 murine macrophages typically have a very short lifespan of about 4-6 days, however MAP infected cell cultures can survive up to about 10 days. Using a modified protocol of Estrella et al. (2011), we have been able to establish a 45-60 day long-term MAP infected J774A.1 murine macrophage cell culture model. With the addition of retinoic acid (RA), vitamin D (VD), and phorbol myristate acetate (PMA) in combination in cell culture, we were able to screen novel therapeutics before embarking on in vivo testing in animals. This is a significant step forward in Crohn's and Johne's disease treatment research. We are not only able to test various drugs against specific strains of MAP to determine susceptibility, but we are also able to test a wide variety of drugs at the same time, with relatively minimal cost. We have evaluated the efficacy of clarithromycin, azithromycin, isoniazid, amikacin, ethambutol, ciprofloxacin, levofloxacin, rifampicin, clofazimine, as well as a combination of clarithromycin, rifampicin, and clofazimine using our MAP infected macrophage cell culture model. We were able to determine the drugs' differential ability to kill intracellular MAP in the early stages of infection, versus chronic stages of infection, and against two different strains of MAP, 43015 and 19698 that affect humans and cattle respectively. The minimal inhibitory concentration (MIC) of each drug was determined as per NCCLS protocol in vitro, and the drugs were tested at the MIC value, along with one concentration above and below the MIC in our cell culture model. The antimicrobials were found to be effective at different stages of cell culture infection and in different strains of MAP. Some drugs were more effective at early stages of MAP infection, whereas others were more effective in chronic or latent stages of infections. It is important to note that although a drug may be effective at a certain stage of infection, it may not necessarily be effective against all strains of MAP. The most promising results were seen with a combination of clarithromycin, clofazimine, and rifampicin, which was effective at all stages of infection with both strains of MAP tested. This long term cell culture model will provide researchers with important screening tools for evaluating new therapeutics before embarking on costly in vivo testing, and allow the assessment of therapeutics at different stages of MAP infection but also against an array of intracellular pathogens. / Ph. D. retinoic acid vitamin D phorbol myristate acetate J774A.1 murine macrophages peptide nucleic acids
4	Mecanismes de regulació en l'activitat biològica del factor de transcripció Snail Domínguez Solà, David 03 April 2003 (has links) Els factors de transcripció de la família Snail són fonamentals en la "transició epiteli-mesènquima", procés morfogènic essencial en el desenvolupament embrionari i en els fenòmens metastàsics tumorals.En els mamífers l'activitat d'Snail és modulada per dos mecanismes. (i) En el promotor humà es troben regions definides de resposta a factors repressors, predominants en les cèl·lules epitelials, i elements diferenciats de resposta a inductors de la "transició epiteli-mesènquima". (ii) L'activitat d'Snail és condicionada també per la seva localització subcel·lular, modulada per mecanismes no transcripcionals: la fosforilació d'Snail determina si és o no exclós del nucli. Al citosol no pot actuar com a repressor transcripcional però pot interaccionar amb la xarxa microtubular, que estabilitza i en condiciona el dinamisme. Això coincideix amb l'activació de la GTPasa RhoA i la reorientació dels filaments de vimentina, fets associats a l'adquisició de capacitat migratòria. L'efecte com a repressor transcripcional i la modulació del dinamisme microtubular són possiblement esdeveniments coordinats necessaris per al rol biològic d'Snail en mamífers. / Snail family of transcription factors is fundamental to the "epithelial-mesenchymal transition", morphogenic process essential to embryonic development and metastatic phenomena in tumors.Snail's activity is modulated in two ways in mammals. (i) The human promoter harbors definite regions that respond to repressor factors, which prevail in epithelial cells; and differentiated elements that respond to known inducers of the "epithelial-mesenchymal transition". (ii) Snail's activity is also conditioned by its subcellular localization, mechanism not dependent on its transcriptional control: Snail phosphorylation determines whether Snail is excluded or not from the nucleus. When in the cytosol, Snail is unable to act as a transcriptional repressor, but however binds to the microtubular meshwork, which becomes stabilized and whose dynamism is conditioned as a result. This fact coincides with the activation of the RhoA GTPase and reorientation of vimentin filaments, both phenomena being related to the acquisition of cell motility. The transcriptional repressor and the microtubule dynamics effects are probably two coordinated events necessary to Snail's biological role in mammals. Polimerització in vitro PKCzeta PKC atípica (Protein Kinase C) P65 Promotor Nocodazol NF-kB/Dorsal (Nuclear Factor Kappa-B) NES (seqüència d'export nuclear) Microtúbuls detirosinats Microtúbuls Metàstasi Invasió Adenocarcinoma ARNm Beta-catenina Centrosoma CLIP-170 Cancer Cresta neural CRM1 (Chromosome Region Maintenance 1) Despolimerització microtúbuls Dit de Zenc atípic Dit de Zenc Domini ric en Serines Ebox E-cadherina Espais intercromatínics Estabilització microtúbuls Export nuclear Filaments intermediaris Fosforilació Gastrulació GFP (Green Fluorescent Protein) Heterocarions ILK (Integrin Linked Kinase) Promotor Reconstrucció fronts invasius Regulació Retrogen RhoA GTPasa SC35 Slug SNAG (domini) SNAI1L Snail Speckles nuclears Time-lapse Èster de forbol Transició Epiteli-Mesènquima Transcripció / Transcripcional U2AF65 Vimentina 57

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