Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for 'disease in a dish' models of muscular physiology and dysfunction

Acute conversion of patient-derived Duchenne muscular dystrophy iPSC into myotubes reveals constitutive and inducible over-activation of TGFβ-dependent pro-fibrotic signaling

Background In Duchenne muscular dystrophy (DMD), DYSTROPHIN deficiency exposes myofibers to repeated cycles of contraction/degeneration, ultimately leading to muscle loss and replacement by fibrotic tissue. DMD pathology is typically exacerbated by excessive secretion of TGFβ and consequent accumulation of pro-fibrotic components of the extra-cellular matrix (ECM), which in turn impairs compensatory regeneration and complicates the efficacy of therapeutic strategies. It is currently unclear whether DMD skeletal muscle fibers directly contribute to excessive activation of TGFβ. Development of skeletal myofibers from DMD patient-derived induced pluripotent stem cells (iPSC), as an “in dish” model of disease, can be exploited to determine the myofiber contribution to pathogenic TGFβ signaling in DMD and might provide a screening platform for the identification of anti-fibrotic interventions in DMD. Methods We describe a rapid and efficient method for the generation of contractile human skeletal muscle cells from DMD patient-derived hiPSC, based on the inducible expression of MyoD and BAF60C (encoded by SMARCD3 gene), using an enhanced version of piggyBac (epB) transposone vectors. DMD iPSC-derived myotubes were tested as an “in dish” disease model and exposed to environmental and mechanical cues that recapitulate salient pathological features of DMD. Results We show that DMD iPSC-derived myotubes exhibit a constitutive activation of TGFβ-SMAD2/3 signaling. High-content screening (HCS)-based quantification of nuclear phosphorylated SMAD2/3 signal revealed that DMD iPSC-derived myotubes also exhibit increased activation of the TGFβ-SMAD2/3 signaling following exposure to either recombinant TGFβ or electrical pacing-induced contraction. Conclusions Acute conversion of DMD patient-derived iPSC into skeletal muscles, by the ectopic expression of MyoD and BAF60C, provides a rapid and reliable protocol for an “in dish” DMD model that recapitulates key pathogenic features of disease pathology, such as the constitutive activation of the TGFβ/SMAD signaling as well as the deregulated response to pathogenic stimuli, e.g., ECM-derived signals or mechanical cues. Thus, this model is suitable for the identification of new therapeutic targets in DMD patient-specific muscles

Assessing physical properties of amphoteric fluoroquinolones using phosphorescence spectroscopy

[EN] The self-association of fluoroquinolones (FQ) in water would play a relevant role in their translocations across lipid membranes. Triplet excited states of these drugs have been shown as reporters of FQ self-association using laser flash photolysis technique. A study using low-temperature phosphorescence technique was performed with quinolone derivatives such as enoxacin (ENX), norfloxacin (NFX), pefloxacin (PFX), ciprofloxacin (CPX, ofloxacin (OFX), nalidixic acid (NLA), pipemidic acid (PPA) and piromidic acid (PRA) to explore emission changes associated with self-associations and to shed some light on the triplet excited state energy (E-T) discrepancies described in the literature for most of these drugs. The emissions obtained at 77 K in buffered aqueous medium revealed that the amphoteric nature of the quinolones CPX, NFX, PFX, ENX, OFX and PPA must generate their self-associations because a redshift of their phosphorescence maxima is produced by FQ concentrations increases. Hence, this effect was not observed for NLA and PRA or when all quinolones were analysed using ethanol or ethylene glycol aqueous mixtures as glassed solvents. Interestingly, the presence of these organic mixtures produced a blue-shift in the phosphorescence emission maximum of each FQ. Additionally, laser flash photolysis experiments with PRA and the amphoteric quinolone PPA, compounds with the same skeleton but different peripheral substituent, confirm the expected correlations between the amphoteric nature of compounds and their self-associations in aqueous media because the excimer generation was only detected for PPA. Now, the discrepancies described in the literature for the ET of FQs can be understood considering that changes of medium polarity or proticity as well as the temperature can considerably modify their ET values. Thereby, low-temperature phosphorescence technique, is an effective way to detect molecular self-associations and surrounding changes in quinolones that opens the possibility to evaluate these effects in other drug families. (C) 2019 Elsevier B.V. All rights reserved.Financial support from Spanish government (grant CTQ2014-54729-C2-2-P) and the Generalitat Valenciana (PROMETEO program, 2017-075).Soldevila Serrano, S.; Bosca Mayans, F. (2020). Assessing physical properties of amphoteric fluoroquinolones using phosphorescence spectroscopy. Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy. 227:1-7. https://doi.org/10.1016/j.saa.2019.117569S17227Domagala, J. M., Hanna, L. D., Heifetz, C. L., Hutt, M. P., Mich, T. F., Sanchez, J. P., & Solomon, M. (1986). New structure-activity relationships of the quinolone antibacterials using the target enzyme. The development and application of a DNA gyrase assay. Journal of Medicinal Chemistry, 29(3), 394-404. doi:10.1021/jm00153a015Cramariuc, O., Rog, T., Javanainen, M., Monticelli, L., Polishchuk, A. V., & Vattulainen, I. (2012). Mechanism for translocation of fluoroquinolones across lipid membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1818(11), 2563-2571. doi:10.1016/j.bbamem.2012.05.027Sun, J., Sakai, S., Tauchi, Y., Deguchi, Y., Chen, J., Zhang, R., & Morimoto, K. (2002). Determination of lipophilicity of two quinolone antibacterials, ciprofloxacin and grepafloxacin, in the protonation equilibrium. European Journal of Pharmaceutics and Biopharmaceutics, 54(1), 51-58. doi:10.1016/s0939-6411(02)00018-8Sun, J., Sakai, S., Tauchi, Y., Deguchi, Y., Cheng, G., Chen, J., & Morimoto, K. (2003). Protonation equilibrium and lipophilicity of olamufloxacin (HSR-903), a newly synthesized fluoroquinolone antibacterial. European Journal of Pharmaceutics and Biopharmaceutics, 56(2), 223-229. doi:10.1016/s0939-6411(03)00099-7Furet, Y. X., Deshusses, J., & Pechère, J. C. (1992). Transport of pefloxacin across the bacterial cytoplasmic membrane in quinolone-susceptible Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 36(11), 2506-2511. doi:10.1128/aac.36.11.2506Maurer, N., Wong, K. F., Hope, M. J., & Cullis, P. R. (1998). Anomalous solubility behavior of the antibiotic ciprofloxacin encapsulated in liposomes: a 1H-NMR study. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1374(1-2), 9-20. doi:10.1016/s0005-2736(98)00125-4Cuquerella, M. C., Andreu, I., Soldevila, S., & Bosca, F. (2012). Triplet Excimers of Fluoroquinolones in Aqueous Media. The Journal of Physical Chemistry A, 116(21), 5030-5038. doi:10.1021/jp301800qLhiaubet-Vallet, V., Sarabia, Z., Boscá, F., & Miranda, M. A. (2004). Human Serum Albumin-Mediated Stereodifferentiation in the Triplet State Behavior of (S)- and (R)-Carprofen. Journal of the American Chemical Society, 126(31), 9538-9539. doi:10.1021/ja048518gBosca, F. (2012). Seeking to Shed Some Light on the Binding of Fluoroquinolones to Albumins. The Journal of Physical Chemistry B, 116(11), 3504-3511. doi:10.1021/jp208930qCuquerella, M. C., Lhiaubet-Vallet, V., Miranda, M. A., & Bosca, F. (2017). Drug–DNA complexation as the key factor in photosensitized thymine dimerization. Physical Chemistry Chemical Physics, 19(7), 4951-4955. doi:10.1039/c6cp08485kAlfredson, T. V., Maki, A. H., & Waring, M. J. (1991). Optically detected triplet-state magnetic resonance studies of the DNA complexes of the bisquinoline analog of echinomycin. Biochemistry, 30(40), 9665-9675. doi:10.1021/bi00104a014Alfredson, T. V., & Maki, A. H. (1990). Phosphorescence and optically detected magnetic resonance studies of echinomycin-DNA complexes. Biochemistry, 29(38), 9052-9064. doi:10.1021/bi00490a024Li, J., Li, J., Shuang, S., & Dong, C. (2005). Study of the luminescence behavior of seven quinolones on a paper substrate. Analytica Chimica Acta, 548(1-2), 134-142. doi:10.1016/j.aca.2005.04.053Sun, C., Ping, H., Zhang, M., Li, H., & Guan, F. (2011). Spectroscopic studies on the lanthanide sensitized luminescence and chemiluminescence properties of fluoroquinolone with different structure. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 82(1), 375-382. doi:10.1016/j.saa.2011.07.065Rieutord, A., Vazquez, L., Soursac, M., Prognon, P., Blais, J., Bourget, P., & Mahuzier, G. (1994). Fluoroquinolones as sensitizers of lanthanide fluorescence: application to the liquid chromatographic determination of ciprofloxacin using terbium. Analytica Chimica Acta, 290(1-2), 215-225. doi:10.1016/0003-2670(94)80058-8Sortino, S., De Guidi, G., Giuffrida, S., Monti, S., & Velardita, A. (1998). pH Effects on the Spectroscopic and Photochemical Behavior of Enoxacin: A Steady-State and Time-Resolved Study. Photochemistry and Photobiology, 67(2), 167. doi:10.1562/0031-8655(1998)0672.3.co;2Martínez, L., Bilski, P., & Chignell, C. F. (1996). Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin. Photochemistry and Photobiology, 64(6), 911-917. doi:10.1111/j.1751-1097.1996.tb01855.xBilski, P., Martinez, L. J., Koker, E. B., & Chignell, C. F. (1996). Photosensitization by Norfloxacin is a Function of pH. Photochemistry and Photobiology, 64(3), 496-500. doi:10.1111/j.1751-1097.1996.tb03096.xBosca, F., Lhiaubet-Vallet, V., Cuquerella, M. C., Castell, J. V., & Miranda, M. A. (2006). The Triplet Energy of Thymine in DNA. Journal of the American Chemical Society, 128(19), 6318-6319. doi:10.1021/ja060651gLhiaubet-Vallet, V., Cuquerella, M. C., Castell, J. V., Bosca, F., & Miranda, M. A. (2007). Triplet Excited Fluoroquinolones as Mediators for Thymine Cyclobutane Dimer Formation in DNA. The Journal of Physical Chemistry B, 111(25), 7409-7414. doi:10.1021/jp070167fCuquerella, M. C., Lhiaubet-Vallet, V., Bosca, F., & Miranda, M. A. (2011). Photosensitised pyrimidine dimerisation in DNA. Chemical Science, 2(7), 1219. doi:10.1039/c1sc00088hLhiaubet-Vallet, V., Bosca, F., & Miranda, M. A. (2009). Photosensitized DNA Damage: The Case of Fluoroquinolones. Photochemistry and Photobiology, 85(4), 861-868. doi:10.1111/j.1751-1097.2009.00548.xAlbini, A., & Monti, S. (2003). Photophysics and photochemistry of fluoroquinolones. Chemical Society Reviews, 32(4), 238. doi:10.1039/b209220bCuquerella, M. C., Miranda, M. A., & Bosca, F. (2006). Role of Excited State Intramolecular Charge Transfer in the Photophysical Properties of Norfloxacin and Its Derivatives. The Journal of Physical Chemistry A, 110(8), 2607-2612. doi:10.1021/jp0559837Lorenzo, F., Navaratnam, S., & Allen, N. S. (2008). Formation of Secondary Triplet Species after Excitation of Fluoroquinolones in the Presence of Relatively Strong Bases. Journal of the American Chemical Society, 130(37), 12238-12239. doi:10.1021/ja804471

Chemical tuning for potential antitumor fluoroquinolones

[EN] Phototoxic effects of 6,8 dihalogenated quinolones confers to this type of molecules a potential property as photochemotherapeutic agents. Two photodehalogenation processes seem to be involved in the remarkable photoinduced cellular damage. In this context, a new 6,8 dihalogenated quinolone 1 (1-methyl-6,8-difluoro-4-oxo-7-aminodimethy1-1,4-dihydroquinoline-3-carboxylic acid) was synthesized looking for improving the phototoxic properties of fluoroquinolones (FQ) and to determine the role of the photodegradation pathways in the FQ phototoxicity. With this purpose, fluorescence emissions, laser flash photolysis experiments and photodegradation studies were performed with compound 1 using 1-ethyl-6,8-difluoro-4-oxo-7-aminodimethy1-1,4-dihidroquinoline-3-carboxylic acid (2) and lomefloxacin (LFX) as reference compounds. The shortening of alkyl chain of the N(1) of the quinolone ring revealed a lifetime increase of the reactive aryl cation generated from photolysis of the three FQ and a significant reduction of the FQ photodegradation quantum yield. The fact that these differences were smaller when the same study was done using a hydrogen donor solvent (ethanol-aqueous buffer, 50/50 v/v) evidenced the highest ability of the reactive intermediate arising from 1 to produce intermolecular alkylations. These results were correlated with in vitro 3T3 NRU phototoxicity test. Thus, when PhotoIrritation-Factor (PIF) was determined for 1, 2 and LFX using cytotoxicity profiles of BALB/c 3T3 fibroblasts treated with each compound in the presence and absence of UVA light, a PIF more higher than 30 was obtained for 1 while the values for 2 and LFX were only higher than 8 and 10, respectively. Thereby, the present study illustrates an approach to modulate the photosensitizing properties of FQ with the purpose to improve the chemotherapeutic properties of antitumor quinolones. Moreover, the results obtained in this study also evidence that the key pathway responsible for the phototoxic properties associated with dihalogenated quinolones is the aryl cation generation.Financial support from Spanish government (MINECO grant CTQ2014-54729-C2-2-P and Severo Ochoa fellowship for C. A., Carlos III Institute of Health grant PI16/01877), and the Generalitat Valenciana (PROMETEO program, 2017-075). We thank M.P. Marin of IIS La Fe Microscopy Unit for confocal microscopy.Anaya-González, C.; Soldevila Serrano, S.; García-Laínez, G.; Bosca Mayans, F.; Andreu Ros, MI. (2019). Chemical tuning for potential antitumor fluoroquinolones. Free Radical Biology and Medicine. 141:150-158. https://doi.org/10.1016/j.freeradbiomed.2019.06.010S150158141Domagala, J. M., Hanna, L. D., Heifetz, C. L., Hutt, M. P., Mich, T. F., Sanchez, J. P., & Solomon, M. (1986). New structure-activity relationships of the quinolone antibacterials using the target enzyme. The development and application of a DNA gyrase assay. Journal of Medicinal Chemistry, 29(3), 394-404. doi:10.1021/jm00153a015Kang, D.-H., Kim, J.-S., Jung, M.-J., Lee, E.-S., Jahng, Y., Kwon, Y., & Na, Y. (2008). New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor. Bioorganic & Medicinal Chemistry Letters, 18(4), 1520-1524. doi:10.1016/j.bmcl.2007.12.053Azéma, J., Guidetti, B., Dewelle, J., Le Calve, B., Mijatovic, T., Korolyov, A., … Kiss, R. (2009). 7-((4-Substituted)piperazin-1-yl) derivatives of ciprofloxacin: Synthesis and in vitro biological evaluation as potential antitumor agents. Bioorganic & Medicinal Chemistry, 17(15), 5396-5407. doi:10.1016/j.bmc.2009.06.053Cullen, M., & Baijal, S. (2009). Prevention of febrile neutropenia: use of prophylactic antibiotics. British Journal of Cancer, 101(S1), S11-S14. doi:10.1038/sj.bjc.6605270Kim, K., Pollard, J. M., Norris, A. J., McDonald, J. T., Sun, Y., Micewicz, E., … McBride, W. H. (2009). High-Throughput Screening Identifies Two Classes of Antibiotics as Radioprotectors: Tetracyclines and Fluoroquinolones. Clinical Cancer Research, 15(23), 7238-7245. doi:10.1158/1078-0432.ccr-09-1964Al-Trawneh, S. A., Zahra, J. A., Kamal, M. R., El-Abadelah, M. M., Zani, F., Incerti, M., … Vicini, P. (2010). Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents. Bioorganic & Medicinal Chemistry, 18(16), 5873-5884. doi:10.1016/j.bmc.2010.06.098Aldred, K. J., Schwanz, H. A., Li, G., Williamson, B. H., McPherson, S. A., Turnbough, C. L., … Osheroff, N. (2015). Activity of Quinolone CP-115,955 Against Bacterial and Human Type II Topoisomerases Is Mediated by Different Interactions. Biochemistry, 54(5), 1278-1286. doi:10.1021/bi501073vPommier, Y., Leo, E., Zhang, H., & Marchand, C. (2010). DNA Topoisomerases and Their Poisoning by Anticancer and Antibacterial Drugs. Chemistry & Biology, 17(5), 421-433. doi:10.1016/j.chembiol.2010.04.012Palumbo, M., Gatto, B., Zagotto, G., & Palù, G. (1993). On the mechanism of action of quinolone drugs. Trends in Microbiology, 1(6), 232-235. doi:10.1016/0966-842x(93)90138-hPaul, M., Gafter-Gvili, A., Fraser, A., & Leibovici, L. (2007). The anti-cancer effects of quinolone antibiotics? European Journal of Clinical Microbiology & Infectious Diseases, 26(11), 825-831. doi:10.1007/s10096-007-0375-4Perrone, C. E. (2002). Inhibition of Human Topoisomerase IIalpha by Fluoroquinolones and Ultraviolet A Irradiation. Toxicological Sciences, 69(1), 16-22. doi:10.1093/toxsci/69.1.16Lhiaubet-Vallet, V., Bosca, F., & Miranda, M. A. (2009). Photosensitized DNA Damage: The Case of Fluoroquinolones. Photochemistry and Photobiology, 85(4), 861-868. doi:10.1111/j.1751-1097.2009.00548.xMarrot, L., Belaïdi, J. P., Jones, C., Perez, P., Meunier, J. R., Riou, L., & Sarasin, A. (2003). Molecular Responses to Photogenotoxic Stress Induced by the Antibiotic Lomefloxacin in Human Skin Cells: From DNA Damage to Apoptosis. Journal of Investigative Dermatology, 121(3), 596-606. doi:10.1046/j.1523-1747.2003.12422.xMeunier, J.-R., Sarasin, A., & Marrot, L. (2002). Photogenotoxicity of Mammalian Cells: A Review of the Different Assays for In Vitro Testing¶. Photochemistry and Photobiology, 75(5), 437. doi:10.1562/0031-8655(2002)0752.0.co;2Martinez, L. J., Li, G., & Chignell, C. F. (1997). Photogeneration of Fluoride by the Fluoroquinolone Antimicrobial Agents Lomefloxacin and Fleroxacin. Photochemistry and Photobiology, 65(3), 599-602. doi:10.1111/j.1751-1097.1997.tb08612.xChignell, C. F., Haseman, J. K., Sik, R. H., Tennant, R. W., & Trempus, C. S. (2003). 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D., Clements, P. J., Pruimboom-Brees, I., Aylott, M., … Krul, C. A. . (2012). The in vivo rat skin photomicronucleus assay: phototoxicity and photogenotoxicity evaluation of six fluoroquinolones. Mutagenesis, 27(6), 721-729. doi:10.1093/mutage/ges038Soldevila, S., & Bosca, F. (2012). Photoreactivity of Fluoroquinolones: Nature of Aryl Cations Generated in Water. Organic Letters, 14(15), 3940-3943. doi:10.1021/ol301694pCuquerella, M. C., Miranda, M. A., & Boscá, F. (2006). Generation of Detectable Singlet Aryl Cations by Photodehalogenation of Fluoroquinolones. The Journal of Physical Chemistry B, 110(13), 6441-6443. doi:10.1021/jp060634dFreccero, M., Fasani, E., Mella, M., Manet, I., Monti, S., & Albini, A. (2008). Modeling the Photochemistry of the Reference Phototoxic Drug Lomefloxacin by Steady-State and Time-Resolved Experiments, and DFT and Post-HF Calculations. Chemistry - A European Journal, 14(2), 653-663. doi:10.1002/chem.200701099Albini, A., & Monti, S. (2003). 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Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma

Original Article

The pancreas taken from the frog (Rana nigromaculata) was fixed in 1% OsO_4 and sliced into ultrathin sections for electron microscopic studies. The following observations were made: 1. A great \u27number of minute granules found in the cytoplasm of a pancreatic cell were called the microsomes, which were divided into two types, the C-microsome and S-microsome. 2. Electron microsopic studies of the ergastoplasm showed that it is composed of the microsome granules and A-substance. The microsomes were seen embedded in the A-substance which was either filamentous or membranous. The membranous structure, which was called the Am-membrane, was seen to form a sac, with a cavity of varying sizes, or to form a lamella. 3. The Am-membrane has close similarity to α-cytomembrane of Sjostrand, except that the latter is rough-surfaced. It was deduced that the Am-membrane, which is smooth-surfaced, might turn into the rough-surfaced α-cytomembrane. 4. There was the Golgi apparatus in the supranuclear region of a pancreatic cell. It consisted of the Golgi membrane, Golgi vacuole and. Golgi vesicle. 5. The mitochondria of a pancreatic cell appeared like long filaments, and some of them were seen to ramify. 6. The membrane of mitochondria, i. e. the limiting membrane, consisted of the Ammembrane. The mitochondria contained a lot of A-substances, as well as the C-microsomes and S-microsomes. When the mitochondria came into being, there appeared inside them chains of granules, which appeared like strips of beads, as the outgrowths of the A-substance and the microsome granules attached to the Am-membrane. They are the so-called cristae mitochondriales. 7. The secretory granules originate in the microsomes. They came into being when the microsomes gradually thickened and grew in size as various substances became adhered to them. Some of the secretory granules were covered with a membrane and appeared like what they have called the intracisternal granule of Palade.It seemed that this was a phenomenon attendant upon the dissolution and liqutefaction of the secretory granule. 8. Comparative studies were made of the ergastoplasm of the pancreatic cells from the frogs in hibernation, the frogs artificially hungered, the frogs which were given food after a certain period of fasting, the frogs to which pilocarpine was given subcutaneously, and the very young, immature frogs. The studies revealed that the ergastoplasm of the pancreatic cells greatly varied in form with the difference in nutritive condition and with different developmental stages of the cell. The change in form and structure occured as a result of transformation of the microsomes and A-substance. The ergastoplasm, even after it has come into being, might easily be inactivated if nutrition is defective. The ergastoplasm is concerned in the secretory mechanism, which is different from the secretory phenomenon of the secretory granules. It would seem that structurally the mitochondria have no direct relation to this mechanism

Genomics and proteomics approaches to the study of cancer-stroma interactions

<p>Abstract</p> <p>Background</p> <p>The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.</p> <p>Methods</p> <p>The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.</p> <p>Results</p> <p>We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (<it>ARID4A</it>, <it>CALR</it>, <it>GNB2L1</it>, <it>RNF10</it>, <it>SQSTM1</it>, <it>USP9X</it>) were validated by real time PCR.</p> <p>Conclusions</p> <p>A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.</p