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Development 134, 2889–2894. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. 2007

Stavridis, M.P., Lunn, J.S., Collins, B.J. and Storey, K.G.

Notes: The authors studied the role of the Erk1/2 signaling pathway during neural specification in mouse embryonic stem (ES) cells. Undifferentiated ES cells express high levels of the pluripotent marker Nanog but do not express fibroblast growth factor (Fgf5), an early marker of differentiation of ES cells, or Sox1, an early neural transcription factor gene. Using quantitative PCR, levels of Nanog, Fgf5 and Sox1 mRNA were quantitated during ES differentiation in the presence and absence of a MEK inhibitor. Prior to quantitative PCR, 1µg of total RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase. By measuring these mRNA levels, the authors determined that inhibition of the Erk1/2 pathway blocked ES differentiation. (3726)

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J. Biol. Chem. 282, 29211–21. A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits. 2007

Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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Am. J. Pathol. 171, 19–31. A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. 2007

Brehm, R., Zeiler, M., Rüttinger, C., Herde, K., Kibschull, M., Winterhager, E., Willecke, K., Guillou, F., Lécureuil, C., Steger, K., Konrad, L., Biermann, K., Failing, K. and Bergmann, M.

Notes: To study the role of connexin42 (cx43) in testis development, the authors generated a conditional cx43 knockout mouse, which lacked the cx43 gene in Sertoli cells. To confirm that the cx43 gene was deleted in these mice, PCR was performed using primers specific to cx43, 1X Colorless GoTaq® Flexi Reaction Buffer, 2mM MgCl2, dNTPs and 0.15µl of GoTaq® DNA Polymerase. Tissue-specific deletion of cx43 was confirmed by amplifying RNA isolated from mouse testis, heart and tail was also confirmed using the same PCR components. (3713)

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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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J. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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Forensic Sci. Int. 170, 68–72. Allele frequency distribution for 21 autosomal STR loci in Bhutan. 2007

Kraaijenbrink, T., van Driem, G.L., Tshering of Gaselô, K. and de Knijff, P.

Notes: The authors determined the allele frequencies for 21 autosomal STR loci in 936 individuals from the Royal Kingdom of Bhutan using the AmpFlSTR® Identifiler® kit, PowerPlex® 16 System and the F13A01, FESFPS, F13B, LPL (FFFL) Multiplex. DNA was extracted from whole blood samples using the Autopure LS® kit and amplified as directed by the manufacturers. Amplified products were detected using an ABI PRISM® 3100 Genetic Analyzer. (3822)

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Forensic Sci. Int. 168, 227–231. Allele frequency distribution for 21 autosomal STR loci in Nepal. 2007

Kraaijenbrink, T., van Driem, G.L., Opgenort, J.R., Tuladhar, N.M. and de Knijff, P.

Notes: Allele frequency data for 21 autosomal loci was studied in 953 unrelated individuals belonging to 12 major Nepalese groups. DNA was extracted from whole blood using the Autopure LS® kit, then amplified using the PowerPlex® 16 System, AmpFlSTR® Identifiler® kit or F13A01, FESFPS, F13B, LPL Multiplex (FFFL) Multiplex. Amplification products were analyzed using the ABI PRISM® 3100 Genetic Analyzer. Several new alleles not reported in the NIST short tandem repeat database were detected in this population. (3811)

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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. 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, 1073–6. Characterization of the variant allele 9.2 of Penta D locus. 2007

Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.

Notes: During casework analysis using the PowerPlex® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

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Proc. Natl. Acad. Sci. USA 104, 10637–10642. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. 2007

Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

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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, 21798–21809. Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed. 2007

Panis, G., Méjean, V. and Ansaldi, M.

Notes: The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System. (3722)

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J. Biol. Chem. 282, 21818–21828. Degradation of hsp70 and other mRNAs in Drosophila via the 5´ 3´ pathway and its regulation by heat shock. 2007

Bönisch, C., Temme, C., Moritz, B. and Wahle, E.

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-32P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq® DNA Polymerase, 1.5mM MgCl2 and 1X Green GoTaq® Flexi Reaction Buffer. (3707)

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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, 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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Am. J. Pathol. 171, 1153-1167. Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy. 2007

Bodó, E., Tobin, D.J., Kamenisch, Y., Bíró, T., Berneburg, M., Funk, W. and Paus, R.

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay. (3746)

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Plant Physiol. 145, 547–558. Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: Evaluation using clethodim. 2007

Yu, Q., Collavo, A., Zheng, M.Q., Owen, M., Sattin, M. and Powles, S.B.

Notes: The authors characterized mutations in the acetyl-coenzyme A carboxylase (ACCase) gene that confer resistance to the herbicide clethodim in the grass weed Lolium rigidum. The ACCase gene was amplified from clethidem-resistant and susceptible plants, then sequenced to identify previously unknown mutations. Amplifications of ACCase were performed using 300ng of genomic DNA and GoTaq® Green Master Mix. The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products directly or from agarose gels. (3721)

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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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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. 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, 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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Microbiology 153, 3023–3033. Expression analysis of extracellular proteins from Phanerochaete chrysosporium grown on different liquid and solid substrates. 2007

Sato, S., Liu, F., Koc, H. and Tien, M.

Notes: The authors characterized expression of extracellular proteins by white-rot fungus, Phanerochaete chrysosporium, grown on wood. Temporal expression of these proteins was monitored by relative quantitative RT-PCR. Two micrograms of total RNA was reversed transcribed using 1µg of Random Primers at 37°C for 1 hour. PCRs with one set of PCR primers were performed using 0.5 units of GoTaq® DNA Polymerase, 1X reaction buffer, 250µM each dNTP, 0.5µM each primer and 1µl of cDNA. PCRs with two sets of PCR primers were performed using 2.5 units of GoTaq® DNA Polymerase, 1.6X reaction buffer, 500µM each dNTP, 0.5µM each primer and 1µl of cDNA. (3708)

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J. Biol. Chem. 282, 14364–14372. Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling. 2007

Tringali, C., Lupo, B., Anastasia, L., Papini, N., Monti, E., Bresciani, R., Tettamanti, G. and Venerando, B.

Notes: The authors transfected the myleoid leukemic cell line K562 with the cytosolic sialidase Neu2. Expression of Neu2 resulted in a significant decrease in mRNA levels for the anti-apoptotic factors Bcl-XL and Bcl-2 as determined by real-time PCR. Reverse transcription was carried out with 1µg of total RNA using the ImProm-II™ Reverse Transcription System and random hexamers. cDNA representing 10ng of total RNA was used in a real-time PCR to quantitate Bcl-XL and Bcl-2 mRNA levels. (3725)

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