The biliprotein phytochrome regulates plant growth and developmental responses to the ambient light environment through an unknown mechanism. Biochemical analyses demonstrate that phytochrome is an ancient molecule that evolved from a more compact light sensor in cyanobacteria. The cyanobacterial phytochrome Cph1 is a light-regulated histidine kinase that mediates red, far-red reversible phosphorylation of a small response regulator, Rcp1 (response regulator for cyanobacterial phytochrome), encoded by the adjacent gene, thus implicating protein phosphorylation-dephosphorylation in the initial step of light signal transduction by phytochrome.

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Tetraploid embryo complementation assay has shown that mouse ES cells alone are capable of supporting embryonic development and adult life of mice. Newly established F 1 hybrid ES cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell-based genetics feasible. Here we report the establishment and characterization of 12 new F1 hybrid ES cell lines and the use of one of the best (G4) in a gain-and loss-of-function genetic study, where the in vivo phenotypes were assessed directly from ES cell-derived embryos. We found the generation of G4 ES cell-derived animals to be very efficient. Furthermore, even after two consecutive rounds of genetic modifications, the majority of transgenic lines retained the original potential of the parental lines; with 10 -40% of chimeras producing ES cell-derived animals/embryos. Using these genetically altered ES cells, this success rate, in most cases, permitted the derivation of a sufficient number of mutants for initial phenotypic analyses only a few weeks after the establishment of the cell lines. Although the experimental design has to take into account a moderate level of uncontrolled damage on ES cell lines, our proofof-principle experiment provides useful data to assist future designs harnessing the power of this technology to accelerate our understanding of gene function.hybrid ͉ tetraploid complementation assay ͉ vasculogenesis ͉ ES cells T he advent of mouse ES cells (1, 2) has revolutionized the genetic approaches addressing gene function. It helped transform the mouse into the ultimate mammalian model system with significant relevance to human biology. Mutating all of the genes in the mouse has become not only feasible but also a substantial international project supported by the European Union, the National Institutes of Health, and Genome Canada (3). Before this effort, thousands of ES cell lines had already been created, each representing a specific mutation, and many more are currently under way. Introduction of these cells back into the mouse through germ-line transmission is a time-, labor-, and cost-intensive endeavor, followed by the tedious task of phenotypic analyses. Therefore, an assay system, which could accelerate the production of mutant embryos or animals and at the same time provide information about in vivo phenotypes for elucidating gene function in normal developmental and pathological processes, would be of major value.Two specific properties of mouse ES cells render them exceptional tools for genetic research: (i) virtually unlimited proliferation capacity and (ii) pluripotent developmental potential. Their proliferation capability leads to the production of large number of cells and therefore the occurrence and the identification of very rare events, such as homologous recombination or gene trap insertion. The pluripotent developmental potential of ES cells allows them to contribute to the germ line when reintroduced into an embryonic environment through chimera formation. However, an additional pro...

(2015) Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemiareperfusion injury, Autophagy, 11:9, 1537-1560, DOI: 10.1080/15548627.2015 Keywords: autophagy, fasting, ischemia-reperfusion, lysosome, myocardial infarctionAbbreviations: CMA, chaperone-mediated autophagy; CQ, chloroquine; GFP, green fluorescent protein; LAD, left anterior descending; LAMP2, lysosomal-associated membrane protein 2; MOI, multiplicity of infection; NRCMs, neonatal rat ventricular cardiac myocytes; q4D, quaque qutra die/every fourth day; qOD, quaque otra die/every other day; TFEB, transcription factor EB; MAP1LC3B (also abbreviated as LC3), microtubule-associated protein 1 light chain 3, isoform B; TTC, triphenyl tetrazolium chloride; LV, left ventricle; AAR, area at risk; WT, wild type.Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from invivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEBmediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fastinginduced autophagy in myocardial ischemia-reperfusion injury.