scholarly journals Novel Assay for Detection of Progesterone Receptor-Interacting Endocrine Disruptors

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A497-A497
Author(s):  
Diana A Stavreva ◽  
Lyuba Varticovski ◽  
Razi Raziuddin ◽  
Gordon L Hager
Keyword(s):  
Progesterone Receptor ◽  
Endocrine Disruptors ◽  
Fluorescent Protein ◽  
Endocrine Disrupting Chemicals ◽  
Binding Activity ◽  
Endocrine Disrupting ◽  
Chimeric Construct ◽  
Agonist And Antagonist ◽  
A Cell ◽  
Green Fluorescent

Abstract The presence of progesterone receptor (PR)-interacting compounds in the environment may have serious health consequences for humans and wildlife, but the methods for their detection and monitoring are limited. Here we report the development and testing of a cell line expressing a chimeric construct containing ligand-binding domain of progesterone receptor and green fluorescent protein-tagged domain of the glucocorticoid receptor (GFP-GR-PR) under tetracycline regulation. Unlike the constitutively nuclear PR, this chimera is cytoplasmic in the absence of the ligand and translocates to the nucleus in response to the hormone or its analogues. The GFP-GR-PR chimera maintains specificity for binding to progesterone and does not cross-react with GR-activating hormones. A concentration- and time-dependent translocation in response to progesterone confirmed picomolar sensitivity for detecting PR ligands. Importantly, the assay can detect both agonist and antagonist activities and thus can be used for screening environmental samples for contamination with endocrine disruptors and for drug development. Using this approach, we screened water samples collected at 23 sites along 2 major rivers in Virginia: Mattaponi and Rappahannock Rivers. We detected a low, but reproducible PR-binding activity in 34.8 % of the sites tested. The calculated progesterone equivalent concentration (EQ) in some of these sites reached ~ 0.8 ng/L. The assay provides an effect-based approach for screening PR-interacting endocrine disrupting chemicals regardless of whether they exert agonist or antagonist activities. Either one could be seriously disruptive for the health of humans and wildlife.

scholarly journals A Cell-Based Method to Detect Agonist and Antagonist Activities of Endocrine-Disrupting Chemicals on GPER

2020 ◽  
Vol 11 ◽  
Author(s):  
Séverine Périan ◽  
Catherine Cerutti ◽  
Christelle Forcet ◽  
Violaine Tribollet ◽  
Jean-Marc Vanacker
Keyword(s):  
Endocrine Disrupting Chemicals ◽  
Endocrine Disrupting ◽  
Agonist And Antagonist ◽  
A Cell

scholarly journals Stabilization of Polar Localization of a Chemoreceptor via Its Covalent Modifications and Its Communication with a Different Chemoreceptor

2005 ◽  
Vol 187 (22) ◽  
pp. 7647-7654 ◽  
Author(s):  
Daisuke Shiomi ◽  
Satomi Banno ◽  
Michio Homma ◽  
Ikuro Kawagishi
Keyword(s):  
Histidine Kinase ◽  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Molecular Assembly ◽  
Covalent Modification ◽  
Polar Localization ◽  
Receptor Localization ◽  
A Cell ◽  
Covalent Modifications ◽  
Green Fluorescent

ABSTRACT In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

scholarly journals The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

2014 ◽  
Vol 14 (3) ◽  
pp. 206-215 ◽  
Author(s):  
Steven Sykes ◽  
Anthony Szempruch ◽  
Stephen Hajduk
Keyword(s):  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Krebs Cycle ◽  
Content Type ◽  
African Trypanosomes ◽  
Atp Production ◽  
Cycle Enzyme ◽  
A Cell ◽  
Direct Localization ◽  
Green Fluorescent

ABSTRACT α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei . The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei . These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei .

scholarly journals Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

2015 ◽  
Vol 113 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Marie-Aude Plamont ◽  
Emmanuelle Billon-Denis ◽  
Sylvie Maurin ◽  
Carole Gauron ◽  
Frederico M. Pimenta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Activation Mechanism ◽  
Photoactive Yellow Protein ◽  
Reversible Binding ◽  
A Cell ◽  
Rapid Labeling ◽  
Green Fluorescent

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

scholarly journals Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic

2020 ◽  
Vol 8 (7) ◽  
pp. 1051 ◽  
Author(s):  
Aleksandar Božić ◽  
Robin C. Anderson ◽  
Tawni L. Crippen ◽  
Christina L. Swaggerty ◽  
Michael E. Hume ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Saccharomyces Boulardii ◽  
Binding Activity ◽  
Intestinal Cells ◽  
E Coli ◽  
Enteric Infections ◽  
Green Fluorescent

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

scholarly journals Epigenetic modifications associated with in utero exposure to endocrine disrupting chemicals BPA, DDT and Pb

2019 ◽  
Vol 34 (4) ◽  
pp. 309-325 ◽  
Author(s):  
Chinonye Doris Onuzulu ◽  
Oluwakemi Anuoluwapo Rotimi ◽  
Solomon Oladapo Rotimi
Keyword(s):  
Endocrine Disruptors ◽  
Endocrine Disrupting Chemicals ◽  
Endocrine System ◽  
Environmental Chemicals ◽  
Epigenetic Mechanisms ◽  
Epigenetic Alterations ◽  
Endocrine Disrupting ◽  
Non Coding Rnas ◽  
Hormone System ◽  
Developmental Windows

Abstract Endocrine disrupting chemicals (EDCs) are xenobiotics which adversely modify the hormone system. The endocrine system is most vulnerable to assaults by endocrine disruptors during the prenatal and early development window, and effects may persist into adulthood and across generations. The prenatal stage is a period of vulnerability to environmental chemicals because the epigenome is usually reprogrammed during this period. Bisphenol A (BPA), lead (Pb), and dichlorodiphenyltrichloroethane (DDT) were chosen for critical review because they have become serious public health concerns globally, especially in Africa where they are widely used without any regulation. In this review, we introduce EDCs and describe the various modes of action of EDCs and the importance of the prenatal and developmental windows to EDC exposure. We give a brief overview of epigenetics and describe the various epigenetic mechanisms: DNA methylation, histone modifications and non-coding RNAs, and how each of them affects gene expression. We then summarize findings from previous studies on the effects of prenatal exposure to the endocrine disruptors BPA, Pb and DDT on each of the previously described epigenetic mechanisms. We also discuss how the epigenetic alterations caused by these EDCs may be related to disease processes.

scholarly journals Effects of endocrine disruptors in the development of the female reproductive tract

2014 ◽  
Vol 58 (2) ◽  
pp. 153-161 ◽  
Author(s):  
Elaine Maria Frade Costa ◽  
Poli Mara Spritzer ◽  
Alexandre Hohl ◽  
Tânia A. S. S. Bachega
Keyword(s):  
Endocrine Disruptors ◽  
Hormone Receptors ◽  
Reproductive Tract ◽  
Polycystic Ovary ◽  
Endocrine Disrupting Chemicals ◽  
Public Health Problem ◽  
Female Reproductive Tract ◽  
Major Public Health Problem ◽  
Endocrine Disrupting ◽  
Environmental Agencies

Environmental agencies have identified a growing number of environmental contaminants that have endocrine disrupting activity, and these can become a major public health problem. It is suggested that endocrine disruptors could account for the higher-than-expected increase in the prevalence of some non-communicable diseases, such as obesity, diabetes, thyroid diseases, and some cancers. Several endocrine Disrupting Chemicals (EDCs), such as pesticides, bisphenol A, phthalates, dioxins, and phytoestrogens, can interact with the female reproductive system and lead to endocrine disruption. Initially, it was assumed that EDCs exert their effects by binding to hormone receptors and transcription factors, but it is currently known that they may also alter the expression of enzymes involved in the synthesis or catabolism of steroids. Biomonitoring studies have identified these compounds in adults, children, pregnant women, and fetuses. Among the diseases of the female reproductive tract associated with EDCs exposure are the following: precocious puberty, polycystic ovary syndrome, and premature ovarian failure. The different populations of the world are exposed to a great number of chemicals through different routes of infection; despite the various available studies, there is still much doubt regarding the additive effect of a mixture of EDCs with similar mechanisms of action.

scholarly journals A Mutation in a Novel Yeast Proteasomal Gene,RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

1998 ◽  
Vol 9 (10) ◽  
pp. 2917-2931 ◽  
Author(s):  
Teresa Rinaldi ◽  
Carlo Ricci ◽  
Danilo Porro ◽  
Monique Bolotin-Fukuhara ◽  
Laura Frontali
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Mitochondrial Morphology ◽  
Nonpermissive Temperature ◽  
Protein Fusion ◽  
In The Wild ◽  
A Cell ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

We report here the functional characterization of an essentialSaccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of thempr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Involvement of a Rab8-like protein of Dictyostelium discoideum, Sas1, in the formation of membrane extensions, secretion and adhesion during development

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2513-2525 ◽  
Author(s):  
Rhonda R. Powell ◽  
Lesly A. Temesvari
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Fluorescent Protein ◽  
Aggregate Size ◽  
Developmental Context ◽  
Cellular Events ◽  
Membrane Protrusions ◽  
A Cell ◽  
Green Fluorescent ◽  
Cell Cell

Establishment of cell–cell adhesions, regulation of actin, and secretion are critical during development. Rab8-like GTPases have been shown to modulate these cellular events, suggesting an involvement in developmental processes. To further elucidate the function of Rab8-like GTPases in a developmental context, a Rab8-related protein (Sas1) of Dictyostelium discoideum was examined, the expression of which increases at the onset of development. Dictyostelium cell lines expressing inactive (N128I mutant) and constitutively active (Q74L mutant) Sas1 as green fluorescent protein (GFP)-Sas1 chimeras were generated. Cells expressing Sas1Q74L displayed numerous actin-rich membrane protrusions, increased secretion, and were unable to complete development. In particular, these cells demonstrated a reduction in adhesion as well as in the levels of a cell adhesion molecule, gp24 (DdCAD-1). In contrast, cells expressing Sas1N128I exhibited increased cell–cell adhesion and increased levels of gp24. Counting factor is a multisubunit signalling complex that is secreted in early development and controls aggregate size by negatively regulating the levels of cell adhesion molecules, including gp24. Interestingly, the Sas1Q74L mutant demonstrated increased levels of extracellular countin, a subunit of counting factor, suggesting that Sas1 may regulate trafficking of counting factor components. Together, the data suggest that Sas1 may be a key regulator of actin, adhesion and secretion during development.

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