GABAA Receptor Subunit Composition Drives Its Sensitivity to the Insecticide Fipronil

Fipronil (FPN) is a worldwide-used neurotoxic insecticide, targeting, and blocking GABAA receptors (GABAARs). Beyond its efficiency on insect GABAARs, FPN causes neurotoxic effects in humans and mammals. Here, we investigated the mode of action of FPN on mammalian α6-containing GABAARs to understand its inhibitory effects on GABA-induced currents, as a function of the synaptic or extrasynaptic localization of GABAARs. We characterized the effects of FPN by electrophysiology using Xenopus oocytes which were microtransplanted with cerebellum membranes or injected with α6β3, α6β3γ2S (synaptic), and α6β3δ (extrasynaptic) cDNAs. At micromolar concentrations, FPN dose-dependently inhibited cerebellar GABA currents. FPN acts as a non-competitive antagonist on ternary receptors. Surprisingly, the inhibition of GABA-induced currents was partial for extra-synaptic (α6β3δ) and binary (α6β3) receptors, while synaptic α6β3γ2S receptors were fully blocked, indicating that the complementary γ or δ subunit participates in FPN-GABAAR interaction. FPN unexpectedly behaved as a positive modulator on β3 homopentamers. These data show that FPN action is driven by the subunit composition of GABAARs—highlighting the role of the complementary subunit—and thus their localization within a physiological synapse. We built a docking model of FPN on GABAARs, which reveals two putative binding sites. This is consistent with a double binding mode of FPN on GABAARs, possibly one being of high affinity and the other of low affinity. Physiologically, the γ/δ subunit incorporation drives its inhibitory level and has important significance for its toxicity on the mammalian nervous system, especially in acute exposure.

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Melatonin Alleviates Hypoxia-Induced Apoptosis of Granulosa Cells by Reducing ROS and Activating MTNR1B–PKA–Caspase8/9 Pathway

Antioxidants ◽

10.3390/antiox10020184 ◽

2021 ◽

Vol 10 (2) ◽

pp. 184

Author(s):

Jing-Li Tao ◽

Xuan Zhang ◽

Jia-Qi Zhou ◽

Cheng-Yu Li ◽

Ming-Hui Yang ◽

...

Keyword(s):

Granulosa Cells ◽

Caspase 3 ◽

Apoptotic Cell ◽

Heme Oxygenase 1 ◽

Inhibitory Effects ◽

Glutathione S Transferase ◽

Competitive Antagonist ◽

Hypoxia Exposure ◽

Induced Apoptosis

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT–MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

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Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽

10.3390/v13061092 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1092

Author(s):

János András Mótyán ◽

Márió Miczi ◽

Stephen Oroszlan ◽

József Tőzsér

Keyword(s):

Amino Acid ◽

Zinc Finger ◽

Cleavage Site ◽

Potential Role ◽

Binding Mode ◽

Interaction Energies ◽

Vitro Assay ◽

Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

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An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

Cell Death and Disease ◽

10.1038/s41419-021-03934-y ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Thao Thi Thanh Nguyen ◽

Masato Shingyoji ◽

Michiko Hanazono ◽

Boya Zhong ◽

Takao Morinaga ◽

...

Keyword(s):

Wild Type ◽

Inhibitory Effects ◽

Cytotoxic Effects ◽

P53 Expression ◽

Nuclear Factor I ◽

Infected Cells ◽

Wild Type P53 ◽

Mdm2 Inhibitor ◽

P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

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Seed Gamma Irradiation of Arabidopsis thaliana ABA-Mutant Lines Alters Germination and Does Not Inhibit the Photosynthetic Efficiency of Juvenile Plants

Dose-Response ◽

10.1177/1559325820979249 ◽

2020 ◽

Vol 18 (4) ◽

pp. 155932582097924

Author(s):

Darya Babina ◽

Marina Podobed ◽

Ekaterina Bondarenko ◽

Elizaveta Kazakova ◽

Sofia Bitarishvili ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Growth Response ◽

Dose Range ◽

Fine Tuning ◽

Plant Tolerance ◽

Inhibitory Effects ◽

Γ Irradiation ◽

Photosynthetic Responses ◽

Mutant Lines

Plant growth response to γ-irradiation includes stimulating or inhibitory effects depending on plant species, dose applied, stage of ontogeny and other factors. Previous studies showed that responses to irradiation could depend on ABA accumulation and signaling. To elucidate the role of ABA in growth and photosynthetic responses to irradiation, lines Col-8, abi3-8 and aba3 -1 of Arabidopsis thaliana were used. Seeds were γ-irradiated using 60Co in the dose range 50-150 Gy. It was revealed that the dose of 150 Gy affected germination parameters of aba3 -1 and Col-8 lines, while abi3-8 line was the most resistant to the studied doses and even showed faster germination at early hours after γ-irradiation at 50 Gy. These results suggest that susceptibility to ABA is probably more important for growth response to γ-irradiation than ABA synthesis. The photosynthetic functioning of 16-day-old plants mainly was not disturbed by γ-irradiation of seeds, and no indication of photosystem II photoinhibition was noticed, revealing the robustness of the photosynthetic system of A. thaliana. Glutathione peroxidase activity and ABA concentrations in plant tissues were not affected in the studied dose range. These results contribute to the understanding of germination and photosynthesis fine-tuning and of mechanisms of plant tolerance to ionizing radiation.

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Amphetamine-Decreased Progesterone and Estradiol Release in Rat Granulosa Cells: The Regulatory Role of cAMP- and Ca2+-Mediated Signaling Pathways

Biomedicines ◽

10.3390/biomedicines9050493 ◽

2021 ◽

Vol 9 (5) ◽

pp. 493

Author(s):

Chung-Yu Chen ◽

Chien-Rung Chen ◽

Chiao-Nan Chen ◽

Paulus S. Wang ◽

Toby Mündel ◽

...

Keyword(s):

Granulosa Cells ◽

Prostaglandin F2α ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Steroidogenic Enzyme ◽

Estradiol Secretion ◽

Basal Release ◽

Ca2 Channels

The purpose of this study is to evaluate the amphetamine effects on progesterone and estradiol production in rat granulosa cells and the underlying cellular regulatory mechanisms. Freshly dispersed rat granulosa cells were cultured with various test drugs in the presence of amphetamine, and the estradiol/progesterone production and the cytosolic cAMP level were measured. Additionally, the cytosolic-free Ca2+ concentrations ([Ca2+]i) were measured to examine the role of Ca2+ influx in the presence of amphetamine. Amphetamine in vitro inhibited both basal and porcine follicle-stimulating hormone-stimulated estradiol/progesterone release, and amphetamine significantly decreased steroidogenic enzyme activities. Adding 8-Bromo-cAMP did not recover the inhibitory effects of amphetamine on progesterone and estradiol release. H89 significantly decreased progesterone and estradiol basal release but failed to enhance a further amphetamine inhibitory effect. Amphetamine was capable of further suppressing the release of estradiol release under the presence of nifedipine. Pretreatment with the amphetamine for 2 h decreased the basal [Ca2+]i and prostaglandin F2α-stimulated increase of [Ca2+]i. Amphetamine inhibits progesterone and estradiol secretion in rat granulosa cells through a mechanism involving decreased PKA-downstream steroidogenic enzyme activity and L-type Ca2+ channels. Our current findings show that it is necessary to study the possibility of amphetamine perturbing reproduction in females.

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Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818618116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10763-10772 ◽

Cited By ~ 10

Author(s):

Bernd R. Gardill ◽

Ricardo E. Rivera-Acevedo ◽

Ching-Chieh Tung ◽

Filip Van Petegem

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Binding Mode ◽

Conformational Switch ◽

Channel Inactivation ◽

Dependent Inactivation ◽

Ef Hand ◽

Inherited Arrhythmia ◽

A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

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The Role of the Nucleus Accumbens Shell in the Mediation of the Reinforcing Properties of a Safety Signal in Free-Operant Avoidance: Dopamine-Dependent Inhibitory Effects of d-amphetamine

Neuropsychopharmacology ◽

10.1038/npp.2013.337 ◽

2013 ◽

Vol 39 (6) ◽

pp. 1420-1430 ◽

Cited By ~ 17

Author(s):

Anushka BP Fernando ◽

Gonzalo P Urcelay ◽

Adam C Mar ◽

Tony A Dickinson ◽

Trevor W Robbins

Keyword(s):

Nucleus Accumbens ◽

Free Operant Avoidance ◽

Safety Signal ◽

Nucleus Accumbens Shell ◽

Inhibitory Effects ◽

Operant Avoidance ◽

Free Operant

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Inhibitory effects of Yangzheng Xiaoji on angiogenesis and the role of the focal adhesion kinase pathway

International Journal of Oncology ◽

10.3892/ijo.2012.1627 ◽

2012 ◽

Vol 41 (5) ◽

pp. 1635-1642 ◽

Cited By ~ 10

Author(s):

WEN G. JIANG ◽

LIN YE ◽

KE JI ◽

NATASHA FREWER ◽

JIAFU JI ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Inhibitory Effects ◽

Kinase Pathway

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The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Microbiology ◽

10.1099/mic.0.28176-0 ◽

2005 ◽

Vol 151 (9) ◽

pp. 2861-2872 ◽

Cited By ~ 33

Author(s):

Marco Ventura ◽

John G. Kenny ◽

Ziding Zhang ◽

Gerald F. Fitzgerald ◽

Douwe van Sinderen

Keyword(s):

3D Structure ◽

Binding Mode ◽

Dna Interaction ◽

Transcriptional Analysis ◽

Amino Acid Residues ◽

Mobility Shift ◽

Bifidobacterium Breve ◽

Gel Mobility Shift ◽

Slot Blot Analysis

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.

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