scholarly journals Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle

2013 ◽  
Vol 2 ◽  
pp. e86 ◽  
Author(s):  
Lindsay M Wallace ◽  
Andrew Moreo ◽  
K Reed Clark ◽  
Scott Q Harper
Keyword(s):  
Reporter Gene ◽  
Mouse Muscle ◽  
Renilla Reniformis ◽  
Dose Dependent

scholarly journals Up-Regulation of Placental Leptin by Human Chorionic Gonadotropin

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 304-313 ◽  
Author(s):  
Julieta L. Maymó ◽  
Antonio Pérez Pérez ◽  
Víctor Sánchez-Margalet ◽  
José L. Dueñas ◽  
Juan Carlos Calvo ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Western Blot ◽  
Reporter Gene ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Promoter Regions ◽  
Leptin Expression ◽  
Dose Dependent

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 μM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 μm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway. Human chorionic gonadotropin induces leptin expression in trophoblastic BeWo cells and placental explants analyzed by western-blot and reporter gene strategy. This effect involves the MAPK signal transduction pathway.

scholarly journals Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site

2002 ◽  
Vol 157 (4) ◽  
pp. 693-702 ◽  
Author(s):  
Jennifer E. Morgan ◽  
Jacqueline G. Gross ◽  
Charles N. Pagel ◽  
Jonathan R. Beauchamp ◽  
Ariberto Fassati ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cells ◽  
Clonal Analysis ◽  
Tumor Formation ◽  
Mouse Strains ◽  
Recipient Site ◽  
Mouse Muscle ◽  
Antiproliferative Agents ◽  
Host Mouse ◽  
Dose Dependent

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

scholarly journals A Synthetic Riboswitch to Regulate Haloarchaeal Gene Expression

2021 ◽  
Vol 12 ◽  
Author(s):  
Johannes Born ◽  
Kerstin Weitzel ◽  
Beatrix Suess ◽  
Felicitas Pfeifer
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Culture Media ◽  
Fold Increase ◽  
Start Codon ◽  
Genetic Circuit ◽  
Genetic Circuits ◽  
Domains Of Life ◽  
Dose Dependent ◽  
Ribosomal Binding Site

In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon Haloferax volcanii. The riboswitch variants A through E and E∗ were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the bgaH reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the mgfp6 reporter gene under PpA promoter control.

scholarly journals Comparison of 7α-methyl-19-nortestosterone effectiveness alone or combined with progestins on androgen receptor mediated-transactivation

Reproduction ◽  
2012 ◽  
Vol 143 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Rocío García-Becerra ◽  
David Ordaz-Rosado ◽  
Gabriela Noé ◽  
Bertha Chávez ◽  
Austin J Cooney ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Reporter Gene ◽  
Gene Transcription ◽  
Detrimental Effect ◽  
Synergistic Effects ◽  
Estrogen Receptor Α ◽  
Male Contraceptive ◽  
Estrogen Activity ◽  
Dose Dependent

7α-methyl-19-nortestosterone (MENT) is an androgen with potent gonadotropin inhibitory activity and prostate-sparing effects. These attributes give MENT advantages over testosterone as a male contraceptive, but, as in the case of testosterone, a partial dose-dependent suppression of spermatogenesis has been observed. Combination of testosterone or MENT with synthetic progestins improves the rate of azoospermia; however, it is unknown whether these combinations affect hormone androgenicity or exert synergistic effects via progestational or androgenic interaction. Herein, using transactivation assays, we examined the ability of MENT alone or combined with several 19-nor-derived synthetic progestins to activate androgen receptor (AR)-dependent gene transcription. In addition, the capability of 7α-methyl-estradiol (7α-methyl-E2), an aromatized metabolite of MENT, to transactivate gene transcription via estrogen receptor α (ERα; ESR1) or ERβ (ESR2) was also investigated. As expected, MENT induced gene transactivation through either the progesterone receptor (PGR) or the AR. MENT was as efficient as progesterone in activating PGR-mediated reporter gene expression, but it was ten times more potent than testosterone and dihydrotestoterone in activating of AR-driven gene expression. The addition of increasing concentrations of other 19-nortestosterone derivatives (norethisterone or levonorgestrel) did not affect, in a significant manner, the ability of MENT to activate AR-dependent reporter gene transcription. The same results were obtained with different cell lines. 7α-Methyl-E2 resulted in potent estrogen activity via both ER subtypes with efficiency similar to natural E2. These results suggest that the addition of 19-nortestosterone-derived progestins, as a hormonal adjuvant in male fertility strategies for effective spermatogenic suppression, does not display any detrimental effect that would interfere with MENT androgenic transcriptional activity.

Electron Microscopic Analysis of Myonecrosis Induced by a Pure Component Isolated From Rattlesnake Venom

1976 ◽  
Vol 34 ◽  
pp. 292-293
Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu
Keyword(s):  
Gel Filtration ◽  
Microscopic Analysis ◽  
The United States ◽  
Exchange Chromatography ◽  
Pure Component ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Mouse Muscle ◽  
Major Health Problem ◽  
Rattlesnake Venom

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.

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