26 research outputs found

    Comparative chromosome painting discloses homologous Segments in distantly related mammals

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    Comparative chromosome painting, termed ZOO-FISH, using DNA libraries from flow sorted human chromosomes 1,16,17 and X, and mouse chromosome 11 discloses the presence of syntenic groups in distantly related mammalian Orders ranging from primates (Homo sapiens), rodents (Mus musculus), even-toed ungulates (Muntiacus muntjak vaginalis and Muntiacus reevesi) and whales (Balaenoptera physalus). These mammalian Orders have evolved separately for 55-80 million years (Myr). We conclude that ZOO-FISH can be used to generate comparative chromosome maps of a large number of mammalian species

    Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFÎČ cell cycle arrest

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    <p>Abstract</p> <p>Background</p> <p>TGFÎČ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFÎČ in disease. We asked how <it>Rb</it>-deficiency would affect responses to TGFÎČ-induced cell cycle arrest.</p> <p>Results</p> <p>Primary hepatocytes isolated from <it>Rb-floxed </it>mice were infected with an adenovirus expressing CRE-recombinase to delete the <it>Rb </it>gene. In control cells treatment with TGFÎČ prevented cells to enter S phase via decreased cMYC activity, activation of P16<sup>INK4A </sup>and P21<sup>Cip </sup>and reduction of E2F activity. In <it>Rb</it>-null hepatocytes, cMYC activity decreased slightly but P16<sup>INK4A </sup>was not activated and the great majority of cells continued cycling. <it>Rb </it>is therefore central to TGFÎČ-induced cell cycle arrest in hepatocytes. However some <it>Rb</it>-null hepatocytes remained sensitive to TGFÎČ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21<sup>Cip1 </sup>and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of <it>p53 </it>and <it>p21</it><sup><it>Cip1</it></sup>. Hepatocytes deficient in <it>p53 or p21</it><sup><it>Cip1 </it></sup>showed diminished growth inhibition by TGFÎČ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21<sup>Cip </sup>and P53 work through the same pathway to regulate G1/S in response to TGFÎČ. In <it>Rb</it>-deficient cells however, <it>p53 </it>but not <it>p21</it><sup><it>Cip </it></sup>deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity.</p> <p>Conclusion</p> <p>The present results show that otherwise genetically normal hepatocytes with disabled <it>p53</it>, <it>p21</it><sup><it>Cip1 </it></sup>or <it>Rb </it>genes respond less well to the antiproliferative effects of TGFÎČ. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRb function, and to a lesser extend P21<sup>Cip1 </sup>and P53 in hepatocytes may represent an additional new mechanism of escape from TGFÎČ-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.</p

    Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

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    In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field
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