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)

Notes: Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR^® Green dye. (3789)

Notes: Klotho knockout mice exhibit a phenotype of precocious aging, organ failure, osteoporosis. Humans with specific Klotho alleles are at increased risk for osteoporosis, atherosclerosis and decreased lifespan. The authors of this study looked at the expression of Klotho in CD4+ lymphocytes in patients suffering from rheumatoid arthritis and age-matched healthy individuals. cDNA was prepared from total RNA isolated from purified CD4+ lymphocytes using the ImProm-II™ Reverse Transcription System. Klotho expression, protein level and activity was decreased in the lymphocytes from the RA patients. (3654)

Notes: The protein Lin-28 is a translational enhancer in differentiating myoblasts; one target of Lin-28 is insulin-like growth factor 2 (IGF-2). The authors showed that Lin-28 increased expression of an IGF-2 reporter construct in vitro. [³⁵S]-methionine-labeled, His-tagged Lin-28 was expressed in the TNT^® Coupled Reticulocyte Lysate System and purified using the MagZ™ Protein Purification System. Increasing amounts of purified Lin-28 protein were added to a TNT^® Coupled Reticulocyte Lysate System reaction containing an IGF-2 luciferase reporter vector, and as a result, luciferase expression was increased up to threefold. Experiments performed with an irrelevant His-tagged protein of equal size and with an equal number of methionine residues confirmed that the increase in luciferase activity was specific to Lin-28. Side-by-side experiments performed with a luciferase reporter vector without the IGF-2 regulatory element did not show increased luciferase activity. (3717)

Notes: The authors developed a new method for analyzing interactions between lipids and lipid transfer proteins, using sterol carrier protein-2 (SCP-2) as a prototype. SCP-2 was expressed with a modified AviTag, consisting of a prolyl-rich semi-rigid hinge linker and the biotinylated AviTag, in E. coli. Biotinylated SCP-2 was purified using the SoftLink™ Soft Release Avidin Resin and an elution buffer containing 5mM biotin. Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry was used to confirm and SCP-2 contained a single biotin moiety at the expected position. Biotinylated SCP-2 was then immobilized on a streptavidin-coated chip, and the on- and off-rate constants of various lipids were determined using surface plasmon resonance. When expressing biotinylated SCP-2, the authors cotransfected E. coli with the SCP-2 expression construct and a plasmid encoding biotin ligase to enhance biotin ligation. (3855)

Notes: These authors identified HSulf-1 as a downregulated gene in ovarian, breast and several other cancer cell lines. To investigate the clinical impact of this downregulation, tissue microarray was used look for associations between HSulf-1 expression levels and clinico-pathological parameters such as tumor histology, grade, hormone receptor status and presence of recurrent disease. HSulf-1 expression levels were determined by RNA in situ hybridization, using probes developed with the Riboprobe^® System. (3616)

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)

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)

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)

Notes: These authors investigated the regulation and function of MAPK-interacting protein kinases 1 and 2 (MNK 1 and 2) and other proteins involved in the MAPK pathway in a panel of breast cancer cells. They use an anti-phospho-ERK antibody produced by Promega against a proprietary immunogen to examine the activation status and amounts of ERKs by Western blot analysis. (3609)

Notes: These authors used a proteomics-based approach to isolate and identify proteins that interact with the estrogen receptor ERα. The protein DBC-1 was identified as a ligand-independent binding partner of ERα. The amino terminus of DBC-1 bound directly to the ERα hormone binding domain in the absence of estradiol. Initial identification of DBC-1 was based on peptide sequence analysis of proteins that coimmunoprecipitated with unliganded ERα. The CheckMate™ Mammalian Two-Hybrid System was then used to confirm the interaction. DBC-1 was fused to the GAL4-DNA-binding domain in the pBIND vector, and ERα was fused to the VP16 activation domain in the pACT vector. These proteins were then expressed independently or together in HeLa cells and their ability to activate transcription of a luciferase reporter controlled by GAL-4-DNA binding sites (pG5luc) was evaluated in both the presence and absence of estradiol. Further analysis with truncated proteins was used to demonstrate that the interaction between DBC-1 and ERα was mediated by the amino terminal part of the DBC-1 protein. (3628)

Notes: The authors cloned and characterized glucokinases (GlcKs) from Trypanosoma cruzi and Leishmania major. Recombinant GlcKs were expressed in E. coli as His₆ fusion proteins and purified using the HisLink™ Protein Purification Resin. Pellets of GlcK-expressing cells were lysed using lysis buffer (100mM Hepes [pH 6.7], 2.0mM MgCl₂, and 10mM imidazole) and a French press, and His₆-tagged protein was purified from the cleared lysate as directed by the manufacturer using an elution buffer (100mM Hepes [pH 7.6], 2.0mM MgCl₂ and 350mM imidazole. (3916)

Notes: The authors cloned pituitary adenylate cyclase-activating polypeptide (PACAP) from lizard (Podarcis sicula) brain. They then isolated total RNA from lizard brain using the SV Total RNA Isolation System and used 4µg of total RNA in a reverse transcription with ImProm-II™ Reverse Transcriptase and oligo(dT)₁₅ primers at 37°C for 1.5 hours. The PACAP cDNA was amplified by PCR, and the resulting PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System prior to sequencing. (3666)

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)

Notes: The authors developed a multilocus sequence typing scheme (MLST) to examine sequence variation and discriminate between Aspergillus fumigatus strains. They also examined the distribution of MAT1-1 and MAT1-2 sexual idiomorphs in 100 clinical and environmental isolates. Sexual idiomorphs were determined using PCR and a reverse primer to both idiomorphs and a forward primer specific to either MAT-1 or MAT-2. PCRs consisted of 2mM MgCl₂, 200µM DNTPs and 2.5 units of GoTaq^® DNA Polymerase. (3714)

Notes: These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor^® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor^® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays. (3673)

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR^® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard^® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard^® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard^® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

Notes: The authors confirmed the interaction of NKX3.1, a prostate-specific homeodomain protein, and topoisomerase I. To determine of this interaction was dependent upon nucleic acid, two types of pull-down assays were performed in the presence of DNase or RNase. For the GST pull-down assay, fragments of topoisomerase I were expressed as GST-fusion proteins, and NKX3.1 was expressed as an [³⁵S]methionine-labeled protein in the TNT^® Quick Coupled Transcription/Translation System. Equimolar amount of GST or GST-topoisomerase I, bound to glutathione sepharose beads, and NKK3.1 were precipitated . In addition, fragments of NKX3.1 were expressed as polyhistidine-tagged proteins and captured using the MagZ™ Binding Particles. Equal molar amounts of NKX3.1 and [³⁵S]methionine-labeled topoisomerase were incubated to examine protein interaction. (3716)

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)

Notes: The authors of this study investigated the role of okadaic acid (OA), a phosphatase inhibitor, in the regulation of the cell cycle using Vicia faba (fava bean) meristem tissue. They used the Kinase-Glo^® Luminescent Kinase Assay to analyze the activity of Histone H1 kinase in OA-treated and untreated meristem. Twenty micromolar histone was used as the substrate and the ATP concentration was 16µM for the assays. (3930)

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)

Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)

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)

Notes: Exploring the possibility of generating a vaccine against Neisseria meningitidis serogroup B (MenB), the authors of this study tested several antigenic sequences in mice for an anti-MenB response. The DNA vaccine was developed using the pCI-neo Mammalian Expression Vector with inserts composed of synthesized double-stranded oligonucleotides that coded for the desired peptides selected using phage display libraries. A leader sequence or a tetanus toxin universal T cell helper epitope sequence or both were inserted at 5’ of the vector multiple cloning region to obtain secretory or T cell helper peptides. Since IFN-γ was helpful as an adjuvant, a second vector was generated using pCI-neo Mammalian Expression Vector encoding murine IFN-γ was used for coimmunization in the DNA vaccine experiments. The plasmids were grown and isolated using a low-endotoxin plasmid purification system and functional peptide was analyzed by FACS. BALB/c mice (5–7 wk old) were immunized with 150µg of purified DNA (70µg of the vectors expressing IFN-γ and 70µg of the immunizing plasmid). An ELISA was used to determine various antibody levels from mouse blood. (3689)

Notes: To understand the post-translational processing of PKDH1 protein, which has a role in human disease, a single full-length cDNA of human PKHD1 was assembled from three overlapping amplimers generated from a human kidney cDNA library. This PKHD1 cDNA was cloned into the pCI Mammalian Expression Vector, then N-terminal FLAG or EGFP tags and a C-terminal 7XMyc tag were added for detection. However, the expression level was too low, and an expression-enhancing beta-globin intronic sequence was added between the construct promoter and PKHD1 cDNA sequence. The pCI-PKHD1 construct was transfected into HEK 293 cells and the protein expression assessed using affinity tag capture and Western blotting. (3758)