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Frontiers in Immunology 2022The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ) show reduced surface...
The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δγγ (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.
Topics: CD3 Complex; Humans; Interleukin-2; Leucine; Phorbol Esters; Receptors, Antigen, T-Cell; Tumor Necrosis Factor-alpha
PubMed: 36119034
DOI: 10.3389/fimmu.2022.978658 -
Journal of Physiology and Biochemistry Feb 2023Allergic asthma is the most common type of asthma. It is characterized by T2 cell-driven inflammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic...
Allergic asthma is the most common type of asthma. It is characterized by T2 cell-driven inflammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic RNAs (Y-RNAs), a variety of non-coding RNAs that are dysregulated in many cancer types, are also differentially expressed in patients with allergic asthma. Their function in the development of the disease is still unknown. We investigated the potential role of RNY3 RNA (hY3) in the T2 cell inflammatory response using the Jurkat cell line as a model. hY3 expression levels were modulated to mimic the upregulation effect in allergic disease. We evaluated the effect of hY3 over cell stimulation and the expression of the T2 cytokine IL13. Total RNA was isolated and retrotranscribed, and RNA levels were assessed by qPCR. In Jurkat cells, hY3 levels increased upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. When transfecting with high levels of hY3 mimic molecules, cell proliferation rate decreased while IL13 mRNA levels increased upon stimulation compared to stimulated control cells. Our results show the effect of increased hY3 levels on cell proliferation and the levels of IL13 mRNA in Jurkat cells. Also, we showed that hY3 could act over other cells via exosomes. This study opens up new ways to study the potential regulatory function of hY3 over IL-13 production and its implications for asthma development.
Topics: Humans; Asthma; Cell Proliferation; Epigenesis, Genetic; Interleukin-13; Lymphocyte Activation; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; RNA, Untranslated
PubMed: 36089628
DOI: 10.1007/s13105-022-00920-6 -
The Effect of Luteolin on the Modulation of Vascular Contractility via ROCK and CPI-17 Inactivation.Biomolecules & Therapeutics Mar 2023In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to...
In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.
PubMed: 36065763
DOI: 10.4062/biomolther.2022.087 -
Phenol-soluble modulin α and β display divergent roles in mice with staphylococcal septic arthritis.Communications Biology Sep 2022Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMβ...
Phenol-soluble modulin α (PSMα) is identified as potent virulence factors in Staphylococcus aureus (S. aureus) infections. Very little is known about the role of PSMβ which belongs to the same toxin family. Here we compared the role of PSMs in S. aureus-induced septic arthritis in a murine model using three isogenic S. aureus strains differing in the expression of PSMs (Newman, Δpsmα, and Δpsmβ). The effects of PSMs on neutrophil NADPH-oxidase activity were determined in vitro. We show that the PSMα activates neutrophils via the formyl peptide receptor (FPR) 2 and reduces their NADPH-oxidase activity in response to the phorbol ester PMA. Despite being a poor neutrophil activator, PSMβ has the ability to reduce the neutrophil activating effect of PSMα and to partly reverse the effect of PSMα on the neutrophil response to PMA. Mice infected with S. aureus lacking PSMα had better weight development and lower bacterial burden in the kidneys compared to mice infected with the parental strain, whereas mice infected with bacteria lacking PSMβ strain developed more severe septic arthritis accompanied with higher IL-6 and KC. We conclude that PSMα and PSMβ play distinct roles in septic arthritis: PSMα aggravates systemic infection, whereas PSMβ protects arthritis development.
Topics: Animals; Arthritis, Infectious; Bacterial Toxins; Mice; NADP; Oxidoreductases; Staphylococcal Infections; Staphylococcus aureus
PubMed: 36065015
DOI: 10.1038/s42003-022-03839-2 -
Biochemistry and Biophysics Reports Sep 2022Human epidermal growth factor receptor isoform D (EGFR; isoform D) is a soluble protein from a 3 kb alternate mRNA transcript that arises from the human EGFR gene....
Human epidermal growth factor receptor isoform D (EGFR; isoform D) is a soluble protein from a 3 kb alternate mRNA transcript that arises from the human EGFR gene. Several studies have identified this circulating isoform of EGFR as a potential diagnostic biomarker for the detection of early stage of cancers. While the expression of the full-length EGFR (isoform A) is regulated by its cognate ligand, EGF, as well as by phorbol myristate acetate (PMA), no studies have examined the factors regulating the expression of EGFR isoform D. In this study, using breast cancer cell lines, we show that the HER receptor ligands, EGF and neuregulin (NRG-1β), as well as the phorbol ester, PMA, can increase the expression of EGFR isoform D, as well as isoform A. Our results, based on measurement of mRNA levels, suggest that EGF induced expression of both isoform A and isoform D occur through a mitogen activated protein kinase (MAPK)-dependent mechanism, and also suggest that protein kinase C is involved in PMA-induced regulation of both isoforms. We also demonstrate that NRG-1β increases isoform A and isoform D expression via the MAPK-dependent pathway, but this regulation occurs independently of phosphatidylinositol 3-kinase/Akt activation. These results suggest that regulation of EGFR isoform A and isoform D expression occur using similar mechanisms. Despite commonalities in the transcriptional regulation of these two EGFR isoforms, the half-lives of these two transcripts is quite different. Moreover, EGFR isoform D, unlike isoform A, is not post-transcriptionally modulated by EGFR activators in the breast cancer cell line MDA-MB-468.
PubMed: 36039113
DOI: 10.1016/j.bbrep.2022.101326 -
Microorganisms Aug 2022This work focused on obtaining fermented oil cake (cotton or ) via macrofungi growth with potential characteristics for animal feed formulations, such as the presence of...
This work focused on obtaining fermented oil cake (cotton or ) via macrofungi growth with potential characteristics for animal feed formulations, such as the presence of extracellular enzymes, bioactive (ergosterol and antioxidants), and detoxification of antinutritional compounds. The concentration of phorbol esters was reduced by four macrofungi in seed cake (JSC) to non-toxic levels. At least two macrofungi efficiently degraded free gossypol in cottonseed cake (CSC). Fermentation with sp. INPA1646 and sp. INPA1696 resulted in increased ergosterol concentrations, antioxidant activity reduction, and high activity of laccases and proteases. Bromatological analysis indicated high crude protein concentrations, with partial solubilization by fungal proteases. Fermented products from sp. and sp. in JSC or CSC can be considered important biological inputs for monogastric and polygastric animal feed.
PubMed: 36014089
DOI: 10.3390/microorganisms10081670 -
International Journal of Molecular... Aug 2022Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the... (Review)
Review
Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca nor pH were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca concentration ([Ca]) and consequential activation of calmodulin linked to gap junction proteins.
Topics: Anesthetics; Anesthetics, Inhalation; Caffeine; Calcium; Calmodulin; Cell Communication; Connexins; Gap Junctions; Halothane; Heptanol; Ion Channels; Isoflurane; Theophylline
PubMed: 36012286
DOI: 10.3390/ijms23169017 -
Journal of Natural Products Aug 2022The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids... (Review)
Review
The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, ), EBC-47 (), EBC-59 (), EBC-83 (), and EBC-177 () were identified. By chemoselective acylation of EBC-46 () and EBC-177 () the natural triesters and and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate () under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in , suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.
Topics: Australia; Croton Oil; Euphorbiaceae; Phorbols; Trees
PubMed: 35973043
DOI: 10.1021/acs.jnatprod.2c00226 -
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 -
Molecules (Basel, Switzerland) Jul 2022Protopine is a substance used for hemostasis with an anti-inflammatory action and is one of the substances that are actively undergoing experiments to confirm their...
Protopine is a substance used for hemostasis with an anti-inflammatory action and is one of the substances that are actively undergoing experiments to confirm their utility as anticancer agents. This study examined the molecular changes in the cellular signaling pathways associated with inflammatory responses in phorbol 12-myristate 13 acetate (PMA)-induced human hepatocellular carcinoma cell line (Hep G2). The inhibition of PMA-induced phosphorylation of I-κB in HepG2, the effect of protopine on the MAPK signals, the inhibition of COX-2 activity, and the inhibition of MMP-9 as a medium of inflammatory response were evaluated by Western blot and qPCR. The effect of protopine on the survival rates in HepG2 cells was evaluated and found to be stable to a processing concentration of up to 40μM. Subsequent Western blot analyses showed that protopine blocks the transfer of the MAPKs cell signals induced by PMA and the transfer of the subunit of the nuclear factor-kappa B (NF-κB) to the nucleolus. Protopine inhibited the kappa alpha (I-κBα) phosphorylation in the cytosol and blocked PMA-induced inflammation via COX-2 activity inhibition. The expression of MMP-9 at the gene and protein levels, which is associated with cell migration and metastasis, was reduced by protopine.
Topics: Anti-Inflammatory Agents; Benzophenanthridines; Berberine Alkaloids; Cyclooxygenase 2; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 9; NF-kappa B; Tetradecanoylphorbol Acetate
PubMed: 35889472
DOI: 10.3390/molecules27144601