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J. Biol. Chem. 282, 10290–10298. Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities. 2007

Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.

Notes: To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the 35S-labelled protein was then used in a GST-pulldown assay. (3692)

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Mol. Endocrinol. Mar. 13, Epub (ahead of print). The micro-RNA miR-206 targets the human estrogen receptor-α, and represses ERα mRNA and protein expression in breast cancer cells. 2007

Adams, B.D., Furneaux, H. and White, B.

Notes: This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells. (3603)

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FASEB J. 21, 1893–1901. Low expression of COX-2, reduced cumulus expansion, and impaired ovulation in SULT1E1-deficient mice. 2007

Gershon, E., Hourvitz, A., Reikhav, S., Maman, E. and Dekel, N.

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)15, 40 units of RNasin® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

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Am. J. Pathol. 171, 1312–1323. Target genes of neuron-restrictive silencer factor are abnormally up-regulated in human myotilinopathy. 2007

Barrachina, M., Moreno, J., Juvés, S., Moreno, D., Olivé, M. and Ferrer, I.

Notes: These authors used chromatin immunoprecipitation to show that neuron-restrictive silencer factor interacts with the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter in U87-MG, DMS53 and HeLa cells. The neuron-restrictive silencing element (NRSE1) of the UCHL1 promoter was amplified using GoTaq® Flexi DNA Polymerase in a 25µl PCR. (3705)

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J. Biol. Chem. 282, 35405-35415. Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth. 2007

Pilecka, I., Patrignani, C., Pescini, R., Curchod, M.L., Perrin, D., Xue, Y., Yasenchak, J., Clark, A., Magnone, M.C., Zaratin, P., Valenzuela, D., Rommel, C. and van Huijsduijnen, R.H.

Notes: To genotype PTPH1 knock-out (KO), heterozygous (HET), and wild type (WT) mice, tail snips were digested overnight with proteinase K and the DNA trapped using the Wizard® SV 96 Genomic DNA Purification System. Genomic DNA was washed using the Wizard® SV Wash Solution and eluted in 200μl of water at 65°C. The protease was inactivated at 95°C and 2μl of DNA was used for PCR. (3739)

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Genome Res. 17, 1286-1295. Domain-wide regulation of gene expression in the human genome. 2007

Gierman, H.J., Indemans, M.H., Koster, J., Goetze, S., Seppen, J., Geerts, D., van Driel, R. and Versteeg, R.

Notes: The authors explored the possibility of a domain-based level of gene expression regulation for chromosomes by integrating the green fluorescent protein (GFP) reporter in 90 different locations in cultured human cells. This integration was accomplished by infecting human embryonic kidney cells (HEK293) with a lentiviral construct carrying the GFP gene under the control of the ubiquitously expressed human phosphoglycerate kinase (PGK), sorting GFP-positive cells by FACS and selecting clones for expansion. Genomic DNA was isolated from the various clones using the Wizard® SV Genomic DNA Purification System and analyzed by PCR or restriction digested for Southern blotting. (3743)

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Proc. Natl. Acad. Sci. USA 104, 12796-12800. Metabolic plasticity during mammalian development is directionally dependent on early nutritional status. 2007

Gluckman PD, Lillycrop KA, Vickers MH, Pleasants AB, Phillips ES, Beedle AS, Burdge GC, Hanson MA.

Notes: To examine how exposure to leptin in utero affects hepatic gene expression and epigenetic status in adulthood, pregnant Wistar rats were fed a standard diet or 30% undernutrition. The three-day-old pups were exposed to saline or leptin for 10 days, weaned and either fed a standard or high-fat diet. On day 170, the rats were sacrificed and tissues snap frozen. Five micrograms of genomic DNA was isolated from rat liver using the Wizard® SV Genomic DNA Purification System. Then 25ng of the purified DNA was digested with methylation-sensitive restriction enzymes, and the glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor alpha (PPARα) fragments were amplified by real-time PCR. (3744)

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J. Biol. Chem. 282, 8380–8392. RSK2 Mediates Muscle Cell Differentiation through Regulation of NFAT3. 2007

Cho, Y.Y., Yao, K., Bode, A.M., Bergen, H.R. 3rd, Madden, B.J., Oh, S.M., Ermakova, S., Kang, B.S., Choi, H.S., Shim, J.H. and Dong, Z.

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to screen for protein-binding partners for RSK2, a ribosomal S6 kinase involved in myoblast differentiation. The RSK2 cDNA was cloned into the pBIND Vector as bait while several transcription factors were amplified by PCR and cloned into the pACT Vector. The bait pBIND-RSK2 construct, the pACT-transcription factors and pG5-luc Vector were transfected into 293 cells at a 1:1:1 molar ratio. To assess the protein interaction, the cells were lysed, and the firefly luciferase activity was normalized to Renilla luciferase activity. The strongest interaction was with NFAT3. (3574)

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Genetics 176, 161–180. Role of the mod(mdg4) common region in homolog segregation in Drosophila male meiosis. 2007

Soltani-Bejnood, M., Thomas, S.E., Villeneuve, L., Schwarz, K.T., Hong, C.S. and McKee, B.D.

Notes: In this paper, the authors studied the common region of mod(mdg4) and its role in homolog conjunction during meiosis. Genomic DNA was isolated from adult Drosophila using the Wizard® Genomic DNA Purification Kit and mutations identified by PCR and sequencing of the introns. (3576)

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Anticancer Res. 27, 3843–8. Pamidronate down-regulates urokinase-type plasminogen activator expression in PC-3 prostate cancer cells. 2007

Iguchi, K. et al.

Notes: In this paper, the authors hypothesized that bisphosphonates, which are used to prevent tumor metastasis, affect expression of urokinasetype plasminogen activator (uPA), which seems to be critical for prostate cancer metastasis. The authors examined the effect of several bisphosphonates on uPA expression in PC-3 cells. Pamidronate treatment resulted in lower uPA mRNA levels. To investigate the cause, the authors created a uPA reporter construct (pGL3-uPA) by cloning the 5′-flanking region of the human uPA gene upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. PC-3 cells were seeded at a density of 3 × 104 cells/well in 24-well culture plates and transfected with 0.5µg of pGL3-uPA and 1ng of the Renilla luciferase phRL-TK Vector using FuGENE® 6 Transfection Reagent. At 48 hours post-transfection, the authors measured reporter activity using the Dual-Luciferase® Reporter Assay System to learn that treatment with 100µM pamidronate inhibited transcription of the uPA gene. (4384)

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Clinica Chimica Acta 381, 171-175. Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: two cases of allele mismatch in the child. 2007

Narkuti, V., Vellanki, R.N., Gandhi, K.P., Doddapaneni, K.K., Yelavarthi, P.D. and Mangamoori, L.N.

Notes: The authors describe two cases of paternity dispute, one with a maternally mismatched allele at the D18S51 locus and a second with a paternally mismatched D18S51 allele. Seventeen autosomal STR loci were analyzed using the PowerPlex® 16 System and AmpFlSTR® Identifiler® kit. Amplifications were performed using a GeneAmp® PCR System 9700, and amplification products were detected using an ABI PRISM® 310 Genetic Analyzer. Y-STR loci and mitochondrial DNA hypervariable regions HV1 and HV2 were also examined. Sequence analysis of the D18S51 locus revealed an expansion of the maternal allele by one repeat unit in one case and an expansion of the paternal allele by two repeat units in the second case. (3809)

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J. Biol. Chem. 282, 9883–94. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. 2007

Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J

Notes: The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

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J. Forensic Sci. 52, 870–3. Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits. 2007

Hill, C.R., Kline, M.C., Mulero, J.J., Lagacé, R.E., Chang, C.W., Hennessy, L.K. and Butler, J.M.

Notes: The authors analyzed 1,308 samples for concordance between the Identifiler® kit, AmpFlSTR® Minifiler™ kit and PowerPlex® 16 System. DNA was isolated from liquid blood using the manual DNA IQ™ System protocol, and STR amplifications were performed as per the manufacturer's recommendations except that reaction volumes were decreased by half. Amplified products were analyzed using an Applied Biosystems 3130xl and POP™-4 or POP™-6 polymer. Twenty seven disconcordant phenotypes were identified between the Minifiler™ and Identifiler® kits, 14 between Minifiler™ and PowerPlex® 16 kits, and 4 between PowerPlex® 16 and Identifiler® kits. (3770)

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J. Biol. Chem. 282, 29847–29854. Differential regulation of vitamin D receptor (VDR) by the p53 family: p73-dependent induction of VDR upon DNA damage. 2007

Kommagani, R., Payal, V. and Kadakia, M.P.

Notes: The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq® Green Master Mix. (3715)

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J. Biomol. Scr. 12, 546–559. Optimization procedure for small interfering RNA transfection in a 384-well format. 2007

Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (3729)

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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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J. Clin. Microbiol. 45, 3316-3322. Evaluation the Invader Assay with the BACTEC MGIT 960 System for prompt isolation and identification of Mycobacteria from clinical specimens. 2007

Ichimura, S., Nagano, M., Ito, N., Shimojima, M., Egashira, T., Miyamoto, C., Ohkusu, K., and Ezaki, T.

Notes: These authors compared standard culture conditions, DNA isolation and analysis (e.g, sequencing) with a liquid culture, DNA isolation and a homogeneous fluorescent detection system for identifying mycobacterial species. The standard DNA extraction began with a loopful (3–mm3 sphere) of bacterial colony grown on Ogawa slants that used glass beads to mechanically disrupt the cells. The resulting lysate was extracted using phenol/chloroform, and DNA purified from the aqueous phase using a robotic liquid handler AGE-96 (Biotec) and the MagneSil® Blood Genomic, Max Yield System. The DNA extractions were used in PCR and sequencing reactions. (3700)

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J. Biol. Chem. 282, 19052–19061. SOX6 suppresses cyclin D1 promoter activity by interacting with beta-catenin and histone deacetylase 1, and its down-regulation induces pancreatic beta-cell proliferation. 2007

Iguchi, H., Urashima, Y., Inagaki, Y., Ikeda, Y., Okamura, M., Tanaka, T., Uchida, A., Yamamoto, T.T., Kodama, T. and Sakai, J.

Notes: Sex-determining Y-box (SOX) 6 is a transcription factor downregulated in obesity-related insulin-resistant animals. The authors examined the interaction between SOX 6 and β-catenin, a protein that modulates cyclin D1 promoter activity. To characterize the physical interaction, in vitro binding assays were performed using GST-fused SOX 6 and deletion mutants of β-catenin, which were expressed as 35S-labeled proteins in the TNT® T7 Quick Coupled Transcription/Translation System. The GST-fusion proteins were bound to MagneGST® particles and allowed to interact with the β-catenin mutants. Purified GST was used as a negative control to determine nonspecific protein binding. The authors were able to identify the protein domains necessary for SOX 6/β-catenin interaction. Similar binding assays were performed with GST-β-catenin and 35S-labeled T-cell factor in the presence or absence of SOX 6 to show that SOX 6 does not interfere with the binding of β-catenin to TCF. (3685)

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Mol. Cell. Endocrinol. 264, 50-60. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. 2007

Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

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Clin. Chem. 53, 1808–1813. Development of a novel immunoassay for the assessment of plasma Gas6 concentrations and their variation with hormonal status. 2007

Clauser, S., Peyrard, S., Gaussem, P., Crespin, M., Emmerich, J., Aiach, M. and Borgel, D.

Notes: To examine the effects of hormones on Gas6, a plasma vitamin K-dependent protein that may play a role in cardiovascular disease, the authors developed an ELISA test for Gas6, which they tested on blood from male and female volunteers. A recombinant Gas6 control was developed by reverse transcribing the full-length human Gas6 mRNA from human umbilical vein endothelial cells, amplifying the cDNA using nested PCR and after restriction digestion, ligating the insert into the EcoRI and XbaI sites of the pCI-neo Mammalian Expression Vector. The full-length construct was confirmed by sequencing and then tested in the Gas6 ELISA. (3688)

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J. Biol. Chem. 282, 10953–10962. Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes. 2007

Ellison, T.I., Eckert, R.L. and MacDonald, P.N.

Notes: While the absence of the Vitamin D receptor (VDR) has profound effects in skin cells, mutation of 25-hydroxyvitamin D 1α-hydroxylase (24OHase), the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis, has little effect on the skin. To determine how VDR may transactivate independent of the 1,25(OH)2D3 ligand, the human 24-hydroxylase promoter was amplified from MCF-7 genomic DNA, digested with XhoI and HindIII and inserted into the pGL3-Basic Vector. Mutations in the proximal and distal vitamin D response elements in the human 24-hydroxylase promoter were introduced using the GeneEditor™ Site-Directed Mutagenesis System. HaCaT cells, primary human fibroblasts or primary human keratinocytes were seeded at a density of 3.2 × 104 cells/well in 12-well plates and transiently transfected with reporter constructs. After 18 hours, the cells were exposed to 1,25(OH)2D3, 9-cis-retinoic acid, ethanol vehicle, or no additive and harvested 24 hours later. The luciferase activity of the cell lysates was measured using the Dual-Luciferase® Reporter Assay System. Five micrograms of RNA purified from mouse keratinocyte and fibroblast cultures was reverse transcribed and amplified for the 24OHase transcripts using the PCR Master Mix. The products were analyzed on ethidium bromide-stained 2% agarose gels. (3695)

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Nucl. Acids Res. 35, 2390–2402. Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance. 2007

Wu, J., Apontes, P., Song, L., Liang, P., Yang, L. and Li, F.

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase® Reporter Assay System. (3697)

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Cancer Res. 67, 1472-1486. Adaptation of energy metabolism in breast cancer brain metastases. 2007

Chen, E.I., Hewel, J., Kreuger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R., and Felding-Habermann, B.

Notes: This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis. (3613)

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Nucl. Acids Res. 35, 1245-1256. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene 2007

Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.

Notes: Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed. (3641)

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J. Appl. Microbiol. 98, 1001-1009. Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes. 2006

Dupont, K., Vogensen, F.K., and Josephsen, J.

Notes: GoTaq® DNA Polymerase was used in PCR to detect Lactococcus lactis phage DNA strains in whey samples. Phage DNA templates were amplified directly from DNase treated and boiled whey samples. For these reactions, the researchers use 0.25µl (1.25 units) of GoTaq® DNA Polymerase for each 50μl reaction. Primers were designed to distinguish various strains of Lactococcus lactis phage receptor-binding protein genes. (3362)

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