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British Journal of Pharmacology Oct 2021Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by...
BACKGROUND AND PURPOSE
Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG.
EXPERIMENTAL APPROACH
We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation.
KEY RESULTS
Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets.
CONCLUSION AND IMPLICATIONS
We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.
Topics: Animals; Glutamic Acid; HEK293 Cells; Humans; Neurosteroids; Pregnanolone; Rats; Sulfates
PubMed: 33988248
DOI: 10.1111/bph.15529 -
BMC Veterinary Research May 2022Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune...
BACKGROUND
Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.
RESULTS
The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.
CONCLUSIONS
Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.
Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Flow Cytometry; Interleukin-17; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Sheep; Tetradecanoylphorbol Acetate
PubMed: 35513847
DOI: 10.1186/s12917-022-03268-7 -
Clinical and Experimental Medicine May 2022Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular...
Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity, poor response to glucocorticoids and exacerbations, the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO), CXCL8, matrix metalloproteinase-9 (MMP-9), granulocyte-monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO, MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology.
Topics: Asthma; Biomarkers; Extracellular Traps; Histones; Humans; Matrix Metalloproteinase 9; Neutrophils; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A
PubMed: 34342773
DOI: 10.1007/s10238-021-00750-8 -
The Biochemical Journal Aug 2022The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations;...
The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC-PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.
Topics: Humans; Melanoma; Phorbol Esters; Phosphorylation; Protein Kinases; p21-Activated Kinases
PubMed: 35969127
DOI: 10.1042/BCJ20220184 -
Frontiers in Immunology 2020Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of...
BACKGROUND
Neutrophil extracellular traps (NETs) are a defense mechanism in which neutrophils cast a net-like structure in response to microbial infection. NETs consist of decondensed chromatin and about 30 enzymes and peptides. Some components, such as neutrophil elastase (NE) and myeloperoxidase (MPO), present antimicrobial but also cytotoxic properties, leading to tissue injury. Many inflammatory diseases are associated with NETs, and their final role has not been identified. Pulmonary surfactant is known to have immunoregulatory abilities that alter the function of adaptive and innate immune cells. The aim of this study was to investigate the hypothesis that natural surfactant preparations inhibit the formation of NETs.
METHODS
The effect of two natural surfactants (Alveofact and Curosurf) on spontaneous and phorbol-12-myristate-13-acetate-induced NET formation by neutrophils isolated by magnetic cell sorting from healthy individuals was examined. NETs were quantitatively detected by absorption and fluorometric-based assays for the NET-specific proteins (NE, MPO) and cell-free DNA. Immunofluorescence microscopy images were used for visualization.
RESULTS
Both surfactant preparations exerted a dose-dependent inhibitory effect on NET formation. Samples treated with higher concentrations and with 30 min pre-incubation prior to stimulation with phorbol-12-myristate-13-acetate had significantly lower levels of NET-specific proteins and cell-free DNA compared to untreated samples. Immunofluorescence microscopy confirmed these findings.
CONCLUSIONS
The described dose-dependent modulation of NET formation ex vivo suggests an interaction between exogenous surfactant supplementation and neutrophil granulocytes. The immunoregulatory effects of surfactant preparations should be considered for further examination of inflammatory diseases.
Topics: Biological Products; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Traps; Granulocytes; Humans; Immunomodulation; Leukocyte Elastase; Neutrophil Activation; Neutrophils; Phospholipids; Pulmonary Surfactants; Tetradecanoylphorbol Acetate
PubMed: 33574811
DOI: 10.3389/fimmu.2020.582895 -
Cell Reports Jun 2023Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic...
Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.
Topics: Animals; Drosophila Proteins; Calmodulin; Presynaptic Terminals; Drosophila; Synaptic Transmission; Synapses; Neuronal Plasticity
PubMed: 37243591
DOI: 10.1016/j.celrep.2023.112541 -
International Journal of Molecular... Dec 2022Breast cancer (BC) is a common female malignancy, worldwide. BC death is predominantly caused by lung metastasis. According to previous studies, Dihydrotanshinone I...
Breast cancer (BC) is a common female malignancy, worldwide. BC death is predominantly caused by lung metastasis. According to previous studies, Dihydrotanshinone I (DHT), a bioactive compound in Bunge (), has inhibitory effects on numerous cancers. Here, we investigated the anti-metastatic effect of DHT on BC, where DHT more strongly inhibited the growth of BC cells (MDA-MB-231, 4T1, MCF-7, and SKBR-3) than breast epithelial cells (MCF-10a). Additionally, DHT repressed the wound healing, invasion, and migration activities of 4T1 cells. In the 4T1 spontaneous metastasis model, DHT (20 mg/kg) blocked metastasis progression and distribution in the lung tissue by 74.9%. DHT reversed the formation of neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13-acetate, as well as ameliorated NETs-induced metastasis. Furthermore, it inhibited Ly6GMpo neutrophils infiltration and H3Cit expression in the lung tissues. RNA sequencing, western blot, and bioinformatical analysis indicated that could modulate DHT acting on lung metastasis inhibition. The study demonstrated a novel suppression mechanism of DHT on NETs formation to inhibit BC metastasis.
Topics: Female; Humans; Extracellular Traps; Breast Neoplasms; Phenanthrenes; Lung Neoplasms; Tetradecanoylphorbol Acetate; Neutrophils
PubMed: 36499502
DOI: 10.3390/ijms232315180 -
Journal of Virology Feb 2023The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch...
The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.
Topics: Humans; Binding Sites; Epstein-Barr Virus Infections; Gene Expression Regulation, Viral; Genome, Viral; Herpesvirus 4, Human; Virus Activation; Virus Latency; RNA-Dependent RNA Polymerase; Cell Line, Tumor; CCCTC-Binding Factor
PubMed: 36744959
DOI: 10.1128/jvi.01894-22 -
International Journal of Molecular... Nov 2022Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized...
Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(-), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.
Topics: Humans; Lipoproteins, HDL; Extracellular Traps; Phospholipids; Endothelial Cells; Lipoproteins, LDL; Tetradecanoylphorbol Acetate
PubMed: 36430470
DOI: 10.3390/ijms232213992 -
Molecules (Basel, Switzerland) Oct 2022is a xerophytic species widely used in Mexico as an ingredient in sweet food and fermented beverages; it is also used in traditional medicine to treat wound pain and...
is a xerophytic species widely used in Mexico as an ingredient in sweet food and fermented beverages; it is also used in traditional medicine to treat wound pain and rheumatic damage, and as a remedy for psoriasis. Among the various extracts and extract fractions that have been evaluated for their anti-inflammatory effects, the acetonic extract (AaAc) and its acetonic (F-Ac) and methanolic (F-MeOH) fractions were the most active in a xylene-induced ear edema model in mice, when orally administered. Four fractions resulting from chemically resolving F-Ac (F1-F4) were locally applied to mice with phorbol 12-myristate 13-acetate (TPA)-induced ear inflammation; F1 inhibited inflammation by 70% and was further evaluated in a carrageenan-induced mono-arthritis model. When administered at doses of 12.5, 25, and 50 mg/kg, F1 reduced articular edema and the spleen index. In addition, it modulated spleen and joint cytokine levels and decreased pain. According to a GC-MS analysis, the main components of F1 are fatty-acid derivatives: palmitic acid methyl ester, palmitic acid ethyl ester, octadecenoic acid methyl ester, linoleic acid ethyl ester, and oleic acid ethyl ester.
Topics: Mice; Animals; Agave; Anti-Inflammatory Agents; Plant Extracts; Fatty Acids; Edema; Carrageenan; Pain; Esters; Phytotherapy
PubMed: 36364031
DOI: 10.3390/molecules27217204