scholarly journals Trypanocidal Activity of Aziridinyl Nitrobenzamide Prodrugs

2010 ◽  
Vol 54 (10) ◽  
pp. 4246-4252 ◽  
Author(s):  
Chris Bot ◽  
Belinda S. Hall ◽  
Noosheen Bashir ◽  
Martin C. Taylor ◽  
Nuala A. Helsby ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Mammalian Host ◽  
Infection Model ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Parasite Life Cycle ◽  
Trypanocidal Activity ◽  
Life Cycle Stages ◽  
Growth Inhibitory ◽  
Mammalian Infection

ABSTRACT The trypanocidal agents nifurtimox and benznidazole both function as prodrugs and must undergo enzyme-mediated activation, a reaction catalyzed by type I nitroreductase (NTR). In the search for new parasitic therapies, we have utilized this finding to investigate whether aziridinyl nitrobenzamide derivatives have activity against bloodstream-form Trypanosoma brucei and Trypanosoma cruzi amastigotes, parasite stages that replicate in the mammalian host. For T. cruzi drug screening, we generated trypanosomes that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of compounds having a 5-(aziridin-1-yl)-2,4-dinitrobenzyl structure was shown to be metabolized by purified T. brucei NTR and when screened against both parasite life cycle stages displayed significant growth-inhibitory properties: the most potent compounds generated 50% inhibitory concentrations of <1 μM. The trypanocidal activity was shown to be NTR specific, since parasites overexpressing this enzyme were hypersensitive to the aziridinyl dinitrobenzyl agents. We conclude that members of the aziridinyl nitrobenzamide class of nitroheterocycles provide new lead structures that have the potential to treat trypanosomal infections.

scholarly journals Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

2013 ◽  
Vol 58 (1) ◽  
pp. 370-377 ◽  
Author(s):  
Andrew A. Voak ◽  
Karin Seifert ◽  
Nuala A. Helsby ◽  
Shane R. Wilkinson
Keyword(s):  
Mammalian Cells ◽  
Leishmania Major ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Flavin Mononucleotide ◽  
Infection Model ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Growth Inhibitory

ABSTRACTMany of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes andLeishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity againstLeishmania major. Biochemical screens using purifiedL. majorNTR (LmNTR) revealed that compounds containing an aziridinyl-2,4-dinitrobenzyl core were effective substrates for the enzyme and showed that the 4-nitro group was important for this activity. To facilitate drug screening against intracellular amastigote parasites, we generated leishmanial cells that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of aziridinyl-2,4-dinitrobenzyl compounds possessing a 5-amide substituent displayed significant growth-inhibitory properties against the parasite, with the most potent agents generating 50% inhibitory concentrations of <100 nM for the intracellular form. This antimicrobial activity was shown to be LmNTR specific sinceL. major NTR+/−heterozygote parasites were slightly resistance to most aziridinyl dinitrobenzyl agents tested. When the most potent leishmanicidal agents were screened against the mammalian cells in which the amastigote parasites were propagated, no growth-inhibitory effect was observed at concentrations of up to 100 μM. We conclude that the aziridinyl nitrobenzamides represent a new lead structure that may have the potential to treat leishmanial infections.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals Monoclonal Antibodies to Intracellular Stages ofCryptosporidium parvumDefine Life Cycle ProgressionIn Vitro

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Georgia Wilke ◽  
Soumya Ravindran ◽  
Lisa Funkhouser-Jones ◽  
Jennifer Barks ◽  
Qiuling Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Life Cycle ◽  
Gastrointestinal Disease ◽  
Epithelial Cell Line ◽  
Type I ◽  
Parasite Life Cycle ◽  
Major Barrier ◽  
Content Type ◽  
Life Cycle Stages

ABSTRACTAmong the obstacles hinderingCryptosporidiumresearch is the lack of anin vitroculture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidiumantibodies and a lack of markers for staging developmental progression. Previously developed antibodies againstCryptosporidiumwere raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supportedC. parvumgrowth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for theCryptosporidiumresearch community and will facilitate future investigation into parasite biology.IMPORTANCECryptosporidiumis a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages ofCryptosporidium. We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation intoCryptosporidiumbiology and help illuminate growth differences between various culture platforms.

scholarly journals The Angiotensin II Type I Receptor-associated Protein, ATRAP, Is a Transmembrane Protein and a Modulator of Angiotensin II Signaling

2003 ◽  
Vol 14 (12) ◽  
pp. 5038-5050 ◽  
Author(s):  
Marco Lopez-Ilasaca ◽  
Xiushi Liu ◽  
Koichi Tamura ◽  
Victor J. Dzau
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Transmembrane Protein ◽  
At1 Receptor ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Receptor Associated Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal

Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1. In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction. ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14–36, 55–77, and 88–108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109–161. Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles. Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane. Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell. Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters. Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation.

scholarly journals Gonadotropin-Releasing Hormone-Mediated Phosphorylation of Estrogen Receptor-α Contributes to fosB Expression in Mouse Gonadotrophs

Endocrinology ◽  
2009 ◽  
Vol 150 (10) ◽  
pp. 4583-4593 ◽  
Author(s):  
Junling Chen ◽  
Beum-Soo An ◽  
Linan Cheng ◽  
Geoffrey L. Hammond ◽  
Peter C. K. Leung
Keyword(s):  
Transcriptional Activation ◽  
Early Response ◽  
Estrogen Receptor Α ◽  
Camp Response Element Binding ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Type I ◽  
Pituitary Cells ◽  
Luciferase Reporter Gene ◽  
Response Element

Abstract Estrogen receptors (ERs) are activated by their ligands as well as signaling pathways that alter ER phosphorylation in response to peptide hormones and growth factors. In pituitary gonadotrophs, GnRHs act via the type I GnRH receptor (GnRHR). Both GnRH subtypes (GnRH-I and -II) activate an estrogen response element (ERE)-driven luciferase reporter gene in LβT2 mouse pituitary cells, and GnRH-I is most potent in this regard. Moreover, antide (a GnRH antagonist) and a GnRHR small interfering RNA (siRNA) abrogate this effect, whereas an ERα antagonist (ICI 182,780) does not. The ERα in LβT2 cells is phosphorylated at Ser118 in the nucleus and at Ser167 in both nucleus and cytoplasm after GnRH treatments and coincided with increased ERα binding to its coactivator, the p300/cAMP response element-binding protein-associated factor (PCAF). Moreover, siRNA-mediated knockdown of PCAF levels attenuated GnRH-induced ERE-luciferase transactivation in these cells. Most importantly, both GnRH subtypes robustly up-regulated expression of the immediate early response gene, fosB, whereas cotreatment with ERα siRNA or PCAF siRNA attenuated this effect. This appears to occur at the transcriptional level because corecruitment of ERα and PCAF to an ERE within the endogenous fosB promoter was increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. These data demonstrate that GnRH-mediated phosphorylation of ERα in mouse LβT2 pituitary cells results in its rapid association with PCAF and the transcriptional activation of fosB, and we demonstrate that this in turn likely activates other genes in pituitary cells including the FSH β-subunit gene.

scholarly journals Transcription Enhancer Factor 1 Binds Multiple Muscle MEF2 and A/T-Rich Elements during Fast-to-Slow Skeletal Muscle Fiber Type Transitions

2003 ◽  
Vol 23 (15) ◽  
pp. 5143-5164 ◽  
Author(s):  
Natalia Karasseva ◽  
Gretchen Tsika ◽  
Juan Ji ◽  
Aijing Zhang ◽  
Xiaoqing Mao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Networks ◽  
Fiber Type ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Transcription Enhancer ◽  
Enhancer Factor

ABSTRACT In adult mouse skeletal muscle, β-myosin heavy chain (βMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload [MOV]). Our previous βMyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (βA/T-rich −269/−258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp βMyHC promoter (β293wt). Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms. By using the βA/T-rich element as bait in a yeast one-hybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific βA/T-rich binding factor. Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the βA/T-rich element is reacted with MOV-plantaris nuclear extract. Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes. In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp βMyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem βA/T-rich elements. These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.

Circ_BMP2K enhances the regulatory effects of miR-455-3p on its target gene SUMO1 and thereby inhibits the activation of cardiac fibroblasts

2020 ◽  
Vol 98 (5) ◽  
pp. 583-590
Author(s):  
Lei Zhang ◽  
Yun-fei Bian ◽  
Rui Bai ◽  
Xiao-sun Song ◽  
Bin Liang ◽  
...  
Keyword(s):  
Myocardial Fibrosis ◽  
Target Gene ◽  
Cardiac Fibroblasts ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Type Iii Collagen ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Regulatory Effects ◽  
And Migration

Research has shown that some circular RNAs (circRNA) are abnormally expressed in the process of myocardial fibrosis, but the mechanism behind this was unknown. In the process of inducing cardiac fibroblast (CF) activation with TGF-β1 or Ang II, the expression of circRNA circ_BMP2K and miR-455-3p were significantly inhibited, whereas the expression of SUMO1 was promoted. The results from our dual luciferase reporter gene assays, RIP assays, and pull-down assays show that miR-455-3p directly binds circ_BMP2K, thereby mutually promoting their expression levels. SUMO1 is a target gene of miR-455-3p, and circ_BMP2K enhances the inhibitory effects of miR-455-3p on the expression of SUMO1. Further study showed that both circ_BMP2K and miR-455-3p inhibited the expression of alpha-SMA as well as type I and type III collagen, whereas SUMO1 promoted their expression, and miR-455-3p inhibitors or overexpression of SUMO1 reversed the effects of circ_BMP2K and miR-455-3p. Circ_BMP2K and miR-455-3p inhibits cell proliferation and migration and promotes the apoptosis of CFs, but SUMO1 has the opposite effects; miR-455-3p inhibitors or overexpression of SUMO1 reverses the effects of circ_BMP2K and miR-455-3p. Thus, circ_BMP2K promotes the expression of miR-455-3p, down-regulates the expression of SUMO1, and finally, inhibits the activation, growth, and migration of CFs. These results could provide important therapeutic targets and a theoretical basis for regulating the process of myocardial fibrosis.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma

2020 ◽  
Vol 23 ◽  
Author(s):  
Zheng Dong ◽  
Qing-Hua Xu ◽  
Yuan-Bin Zhu ◽  
Yong-Feng Wang ◽  
Jie Xiong ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Lung Squamous Cell Carcinoma ◽  
Luciferase Reporter ◽  
Control Group ◽  
Vascular Endothelial ◽  
Luciferase Reporter Gene ◽  
Endothelial Growth Factor

Aims : The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). Methods: A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment was selected. The expression of miR-22 was detected by fluorescence quantitative real-time PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGF-D) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. Results: The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 cotransfection group reversed the changes. Conclusion: We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

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