Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Leukemic cell lines such as Mono Mac 6 provide an excellent model for studying changes in gene expression during induction of cell differentiation. Mono Mac 6 cells can be induced to differentiate from their immature state to cells resembling morphologically and functionally mature monocytes and macrophages by various stimuli such as calcitriol and transforming growth factor-beta. During differentiation, the expression of differentiation markers such as the cell surface antigen CD14 or other differentiation-related genes such as 5-lipoxygenase are strongly increased. Thus, this cell line constitutes an excellent model system to study the regulation of gene expression by inducers of cell differentiation. However, myeloid cell lines are often refractory to transfection by calcium phosphate or DEAE dextran so that reporter gene assays are difficult to perform. We have established a transient transfection protocol for Mono Mac 6 cells using electroporation, a 5-lipoxygenase promoter luciferase reporter gene construct, and the secreted alkaline phosphatase as an internal standard.
 ...
PMID:Transient transfection of the human myeloid cell line Mono Mac 6 using electroporation. 1254 51

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.
 ...
PMID:Green fluorescent protein as a molecular marker in microbiology. 1258 95

Gene electrotranfer is an attractive physical method to deliver genes to target tissues. The aim of this study was to evaluate in vivo gene electrotransfer into spleen, one of the most important lymphoid organ, in order to create a new tool to modulate the immuno-inflammatory system. C57Bl/6 mice were submitted either to intramuscular electrotransfer (IME) as a reference method or to intrasplenic (ISE) gene electrotransfer. In the naked injected plasmids, the CMV promoter controlled the expression of luciferase, secreted alkaline phosphatase, EGFP, or IFNgamma. The ISE optimal electrotransfer conditions were first determined and ISE was found to be an efficient gene transfer method, which can be used to express secreted or intracellular proteins transiently. Although transfected cells were still present in the spleen 30 days after ISE, transfected spleen cells could recirculate since they were detected in extrasplenic locations. Using a T-lymphocyte-specific promoter controlling the expression of EGFP, splenic T cells could be targeted. Finally, it appeared that ISE procedure does not impair by itself the immune response and does not result in a significant production of antibodies directed to the transgenic proteins in C57Bl/6 mice. This strategy constitutes a new method to manipulate the immune response that can be used in various experimental designs.
 ...
PMID:Non-viral gene transfer of murine spleen cells achieved by in vivo electroporation. 1264 62

This paper describes a novel bioluminescent assay of alkaline phosphatase (ALP) utilizing ATP-sulfurylase and the luciferin-luciferase reaction. The principle governing the assay is as follows. Adenosine-3'-phosphate-5'-phosphosulfate, which serves as the substrate for ALP, is hydrolyzed enzymatically to produce adenosine-5'-phosphosulfate (APS). APS is converted into ATP by ATP-sulfurylase in the presence of pyrophosphate. The ATP produced is detected by the luciferin-luciferase reaction. The measurable range was 1 zmol to 100 fmol/assay and the detection limit at blank+3 SD was 10 zmol/assay. The coefficient of variation (CV, n=5) was examined at each point of the standard curve; the mean CV percentage was 4.47% (n=6). This assay system was applied to enzyme immunoassay of human chorionic gonadotropin and allele-specific PCR enzyme-linked immunosorbent assay of verotoxin gene using ALP as the label enzyme; 10(-2) mIU/mL hCG in urine and 5 pg of Escherichia coli O157 DNA could be assayed directly and with high sensitivity by the proposed method.
 ...
PMID:Novel bioluminescent assay of alkaline phosphatase using adenosine-3'-phosphate-5'-phosphosulfate as substrate and the luciferin-luciferase reaction and its application. 1265 6

It is well established that core binding factor Runx2/Cbfa1 is required for osteoblast recruitment and differentiation from mesenchymal stem cells. Transcriptional regulation of the Runx2/Cbfa1 gene by osteogenic factors such as bone morphogenetic proteins (BMPs) plays an important role in the stimulation of bone formation by these cytokines. BMP7 (also termed OP-1) is a member of the transforming growth factor beta (TGF-beta) superfamily and induces osteoblast differentiation from mesenchymal precursor stem cells in vitro as well as bone formation in vivo. This study examines the effects of BMP7 on markers of osteoblast differentiation and specifically on human Runx2/Cbfa1 gene transcription in a mouse C2C12 myoblast cell line where it induces expression of both alkaline phosphatase (ALP) and endogenous Runx2/Cbfa1. To further understand the mechanisms of human Runx2/Cbfa1 transcriptional regulation by BMP7, we cloned 3.0 kb of the human Runx2/Cbfa1 gene 5'-upstream flanking region and created a series of promoter deletions cloned into luciferase-based reporter vectors (Runx2/Cbfa1/Luc). Sequence data revealed six copies of the osteoblastic cis-acting element (OSE2) in the proximal promoter region. In C2C12 cells transiently transfected with Runx2/Cbfa1/Luc deletion constructs, transcriptional activity of Runx2/Cbfa1 was upregulated up to 2-fold after 24 h of BMP7 treatment. Mutational analysis demonstrated that the minimal responsive promoter region for BMP7-regulated transcription maps to a proximal -74 OSE2 site. Electromobility shift assays with C2C12 cellular extracts indicate that BMP7 increases binding of OSE2 promoter sequences, and supershift assays with anti-Runx2/Cbfa1 antibodies demonstrate that Runx2/Cbfa1 is part of the nucleoprotein complex binding OSE2. Together, these data indicate BMP7 can upregulate Runx2/Cbfa1 gene expression in C2C12 myoblast cells, and suggest that Runx2/Cbfa1 may bind to OSE2 elements within its own promoter to autoregulate gene transcription in differentiating osteoblasts.
 ...
PMID:Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7. 1289 May 74

Black cohosh is an increasingly popular alternative to estrogen replacement therapy for the relief of menopausal symptoms, primarily hot flushes. However, an important consideration for long-term therapy is potential toxicity and carcinogenicity. Therefore, we undertook a study to assess the estrogenic activity of black cohosh to examine its safety for those with, or at high risk of developing, breast cancer. Several assays were utilized as listed: RNAse protection assays, which ascertain the regulation of the expression of E2-responsive genes; estrogen-responsive-element (ERE)-luciferase, which determines modulation of the ER function by transactivation of the ERE; the Ishikawa cell system, which has an E2-regulated endogenous alkaline phosphatase; and colony formation of ER-expressing breast cancer cells, which indicates possible progression of early stage breast cancer into a more aggressive state. Black cohosh extracts did not demonstrate estrogenic activity in any of these assay systems. This is an encouraging step in the assessment of the safety of black cohosh for treatment of menopausal hot flushes.
 ...
PMID:Black cohosh, a menopausal remedy, does not have estrogenic activity and does not promote breast cancer cell growth. 1453 83

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.
 ...
PMID:Characterization of a new class of selective nonsteroidal progesterone receptor agonists. 1507 22

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.
 ...
PMID:5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes. 1510 58

In determining the biological profiles of various antiprogestins, it is important to assess the hormonal and antihormonal activity, selectivity, and potency of their proximal metabolites. The early metabolism of mifepristone is characterized by rapid demethylation and hydroxylation. Similar initial metabolic pathways have been proposed for CDB-2914 (CDB: Contraceptive Development Branch of NICHD) and CDB-4124, and their putative metabolites have been synthesized. We have examined the functional activities and potencies, in various cell-based assays, and relative binding affinities (RBAs) for progesterone receptors (PR) and glucocorticoid receptors (GR) of the putative mono- and didemethylated metabolites of CDB-2914, CDB-4124, and mifepristone and of the 17alpha-hydroxy and aromatic A-ring derivatives of CDB-2914 and CDB-4124. The binding affinities of the monodemethylated metabolites for rabbit uterine PR and human PR-A and PR-B were similar to those of the parent compounds. Monodemethylated mifepristone bound to rabbit thymic GR with higher affinity than monodemethylated CDB-2914 or CDB-4124. T47D-CO cells were used to assess inhibition of R5020-stimulated endogenous alkaline phosphatase activity and transactivation of the PRE(2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid in transient transfections. The antiprogestational potency was as follows: mifepristone/CDB-2914/CDB-4124/monodemethylated metabolites (IC(50)'s approximately 10(-9)M) > aromatic A-ring derivatives (IC(50)'s approximately 10(-8)M) > didemethylated/17alpha-hydroxy derivatives (IC(50)'s approximately 10(-7)M). Antiglucocorticoid activity was determined by inhibition of dexamethasone-stimulated transcriptional activity in HepG2 cells. The mono- and didemethylated metabolites of CDB-2914 and CDB-4124 had less antiglucocorticoid activity (IC(50)'s approximately 10(-6)M) than monodemethylated mifepristone (IC(50) approximately 10(-8)M) or the other test compounds. At 10(-6)M in transcription assays, none of these compounds showed progestin agonist activity, whereas mifepristone and its monodemethylated metabolite manifested slight glucocorticoid agonist activity. The reduced antiglucocorticoid activity of monodemethylated CDB-2914 and CDB-4124 was confirmed in vivo by the thymus involution assay in adrenalectomized male rats. The aromatic A-ring derivatives-stimulated transcription of an estrogen-responsive reporter plasmid in MCF-7 and T47D-CO human breast cancer cells but were much less potent than estradiol. Taken together, these data suggest that the proximal metabolites of mifepristone, CDB-2914, and CDB-4124 contribute significantly to the antiprogestational activity of the parent compounds in vivo. Furthermore, the reduced antiglucocorticoid activity of CDB-2914 and CDB-4124 compared to mifepristone in vivo may be due in part to decreased activity of their putative proximal metabolites.
 ...
PMID:In vitro antiprogestational/antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone. 1512 Apr 21

The transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMP) are key regulatory factors that affect many critical cellular events in growth and development. Recently, we have shown that the Ski-interacting protein (SKIP) can augment TGF-beta signals. Here, we extended these studies by examining the biologic consequences of SKIP overexpression on TGF-beta1 and BMP-2 signals in C2C12 cells. C2C12 myoblasts differentiate into myotubes when the media is depleted of mitogenic factors, and TGF-beta1 inhibits this myotube formation. BMP-2 not only inhibits the myotube formation, but also induces C2C12 cells to differentiate into osteoblasts. Here, we show that SKIP-overexpressing C2C12 cells treated with TGF-beta1 or BMP-2 displayed no differences in comparison to vector control cells in their ability to form myotubes or in the expression of the myogenic markers myosin heavy chain-1 and myogenin. Unexpectedly, SKIP-overexpressing C2C12 cells treated with BMP-2 displayed suppressed expression of the induced osteoblast markers alkaline phosphatase, osteocalcin, and the transcription factor Runx2. Lastly, SKIP could repress transcription induced by BMP-2 in luciferase reporter assays done in C2C12 cells. These data show that SKIP has specific inhibitory effects on BMP-2-induced differentiation and implicate SKIP to be a novel regulator of the differentiation programming induced by TGF-beta signals.
 ...
PMID:Differential effects of the Ski-interacting protein (SKIP) on differentiation induced by transforming growth factor-beta1 and bone morphogenetic protein-2 in C2C12 cells. 1514 47


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>