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Author (up) Bhattacharya, R.; Beck, D.J. file  url
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  Title Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II, RecBCD and RecFOR functions Type Journal Article
  Year 2002 Publication DNA Repair Abbreviated Journal DNA Repair (Amst)  
  Volume 1 Issue 11 Pages 955-966  
  Keywords Antineoplastic Agents/*pharmacology; Bacterial Proteins/physiology; Cell Division/drug effects/genetics/radiation effects; Cisplatin/*pharmacology; DNA Damage/drug effects/radiation effects; DNA Polymerase II/*physiology; DNA Polymerase III/physiology; DNA Repair/drug effects/radiation effects; DNA-Binding Proteins/physiology; Dose-Response Relationship, Drug; Drug Resistance, Bacterial/physiology; Escherichia coli/*drug effects/enzymology; Escherichia coli Proteins/pharmacology/*physiology; Exodeoxyribonuclease V; Exodeoxyribonucleases/*physiology; Lac Operon; SOS Response (Genetics)/*physiology; beta-Galactosidase/metabolism  
  Abstract Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA. The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance. In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair. In this work, the roles of Pol II and HR in facilitating resistance of E. coli to cisplatin are studied. SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E. coli PQ30 that has the lacZ gene fused to the sfiA promoter. Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR. Resistance of E. coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB. Both the RecBCD and RecFOR pathways of HR are important for survival when E. coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants. Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment. In contrast, Pol II was not required for survival in recB mutants. SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way. An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.  
  Call Number Serial 407  
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Author (up) Hellman, L.M.; Fried, M.G. file  url
openurl 
  Title Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions Type Journal Article
  Year 2007 Publication Nature Protocols Abbreviated Journal Nat Protoc  
  Volume 2 Issue 8 Pages 1849-1861  
  Keywords Bacterial Proteins/metabolism; Cyclic AMP Receptor Protein/metabolism; DNA-Binding Proteins/*analysis/metabolism; Electrophoretic Mobility Shift Assay/*methods; Escherichia coli/genetics/metabolism; Escherichia coli Proteins/metabolism; Humans; Lac Operon/genetics; Lac Repressors; O(6)-Methylguanine-DNA Methyltransferase/metabolism; Promoter Regions, Genetic; RNA-Binding Proteins/*analysis; Repressor Proteins/metabolism; Transcription Factors/metabolism  
  Abstract The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.  
  Call Number Serial 1994  
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Author (up) Lambert, G.; Kussell, E. file  url
doi  openurl
  Title Memory and fitness optimization of bacteria under fluctuating environments Type Journal Article
  Year 2014 Publication PLoS Genetics Abbreviated Journal PLoS Genet  
  Volume 10 Issue 9 Pages e1004556  
  Keywords *Adaptation, Biological; Algorithms; *Bacterial Physiological Phenomena; *Environment; Gene Expression Regulation, Bacterial; Lac Operon; Models, Biological; Phenotype; Stress, Physiological  
  Abstract Bacteria prudently regulate their metabolic phenotypes by sensing the availability of specific nutrients, expressing the required genes for their metabolism, and repressing them after specific metabolites are depleted. It is unclear, however, how genetic networks maintain and transmit phenotypic states between generations under rapidly fluctuating environments. By subjecting bacteria to fluctuating carbon sources (glucose and lactose) using microfluidics, we discover two types of non-genetic memory in Escherichia coli and analyze their benefits. First, phenotypic memory conferred by transmission of stable intracellular lac proteins dramatically reduces lag phases under cyclical fluctuations with intermediate timescales (1-10 generations). Second, response memory, a hysteretic behavior in which gene expression persists after removal of its external inducer, enhances adaptation when environments fluctuate over short timescales (< 1 generation). Using a mathematical model we analyze the benefits of memory across environmental fluctuation timescales. We show that memory mechanisms provide an important class of survival strategies in biology that improve long-term fitness under fluctuating environments. These results can be used to understand how organisms adapt to fluctuating levels of nutrients, antibiotics, and other environmental stresses.  
  Call Number Serial 1140  
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Author (up) Sirma, H.; Kumar, M.; Meena, J.K.; Witt, B.; Weise, J.M.; Lechel, A.; Ande, S.; Sakk, V.; Guguen-Guillouzo, C.; Zender, L.; Rudolph, K.-L.; Gunes, C. file  url
doi  openurl
  Title The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation Type Journal Article
  Year 2011 Publication Gastroenterology Abbreviated Journal Gastroenterology  
  Volume 141 Issue 1 Pages 326-37, 337.e1-3  
  Keywords Animals; Binding Sites; Cell Differentiation; *Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; E2F2 Transcription Factor/metabolism; E2F7 Transcription Factor/metabolism; Gene Expression Regulation, Enzymologic; Genes, Reporter; Hepatectomy; Hepatocytes/*enzymology; Humans; Lac Operon; Liver/*enzymology/surgery; *Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; *Promoter Regions, Genetic; RNA Interference; Regulatory Elements, Transcriptional; Retinoblastoma Protein/metabolism; Telomerase/*genetics/metabolism; Time Factors; *Transcriptional Activation  
  Abstract BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.  
  Call Number Serial 430  
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Author (up) Xu, W.; Chi, L.; Xu, R.; Ke, Y.; Luo, C.; Cai, J.; Qiu, M.; Gozal, D.; Liu, R. file  url
openurl 
  Title Increased production of reactive oxygen species contributes to motor neuron death in a compression mouse model of spinal cord injury Type Journal Article
  Year 2005 Publication Spinal Cord Abbreviated Journal Spinal Cord  
  Volume 43 Issue 4 Pages 204-213  
  Keywords Animals; Apoptosis/physiology; Blotting, Western/methods; Caspase 3; Caspases/metabolism; Cell Count/methods; Cytochromes c/metabolism; DNA, Single-Stranded/metabolism; Disease Models, Animal; Female; Guanine/*analogs & derivatives/metabolism; Immunohistochemistry/methods; In Situ Nick-End Labeling/methods; Lac Operon/physiology; Lipid Peroxidation/physiology; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Molecular; Motor Neurons/*pathology; NF-kappa B/genetics; Peroxidases; Proto-Oncogene Proteins c-fos/metabolism; Reactive Oxygen Species/*metabolism; Spinal Cord Injuries/genetics/*metabolism/*pathology/physiopathology; Staining and Labeling/methods; Superoxide Dismutase/genetics; Superoxide Dismutase-1; Time Factors  
  Abstract STUDY DESIGN: Experimental laboratory investigation of the role and pathways of reactive oxygen species (ROS)-mediated motor neuron cell death in a mouse model of compression spinal cord injury. OBJECTIVES: To analyze ROS-mediated oxidative stress propagation and signal transduction leading to motor neuron apoptosis induced by compression spinal cord injury. SETTING: University of Louisville Health Science Center. METHODS: Adult C57BL/6J mice and transgenic mice overexpressing SOD1 were severely lesioned at the lumbar region by compression spinal cord injury approach. Fluorescent oxidation, oxidative response gene expression and oxidative stress damage markers were used to assay spinal cord injury-mediated ROS generation and oxidative stress propagation. Biochemical and immunohistochemical analyses were applied to define the ROS-mediated motor neuron apoptosis resulted from compression spinal cord injury. RESULTS: ROS production was shown to be elevated in the lesioned spinal cord as detected by fluorescent oxidation assays. The early oxidative stress response markers, NF-kappaB transcriptional activation and c-Fos gene expression, were significantly increased after spinal cord injury. Lipid peroxidation and nucleic acid oxidation were also elevated in the lesioned spinal cord and motor neurons. Cytochrome c release, caspase-3 activation and apoptotic cell death were increased in the spinal cord motor neuron cells after spinal cord injury. On the other hand, transgenic mice overexpressing SOD1 showed lower levels of steady-state ROS production and reduction of motor neuron apoptosis compared to that of control mice after spinal cord injury. CONCLUSION: These data together provide direct evidence to demonstrate that the increased production of ROS is an early and likely causal event that contributes to the spinal cord motor neuron death following spinal cord injury. Thus, antioxidants/antioxidant enzyme intervention combined with other therapy may provide an effective approach to alleviate spinal cord injury-induced motor neuron damage and motor dysfunction.  
  Call Number Serial 2145  
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