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Journal of Neurochemistry Dec 2022The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action...
The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release >80% of IRP, are related with a fast dynamin-independent, Ca - and protein kinase C (PKC)-dependent endocytosis (time constant <1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI, PKC 19-31 peptide, and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ~8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.
Topics: Mice; Animals; Protein Kinase C; Patch-Clamp Techniques; Calcium; Exocytosis; Endocytosis; Dynamins
PubMed: 36254753
DOI: 10.1111/jnc.15710 -
Clinical Interventions in Aging 2022The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PURPOSE
The aim of the paper is to establish and quantify the relation between healthy ageing and the innate and adaptive immune parameters as indicators of age-related diseases.
PATIENTS
In order to observe the immunological changes that occur according to age, several humoral and cellular immune parameters were investigated for 288 healthy donors (30-80 years). Subjects' selection was done using clinical, biochemical and immunological parameters of inclusion/exclusion criteria from SENIEUR protocol.
RESULTS
Age-related changes were observed for both humoral and cellular immune parameters. Lymphocyte immunophenotyping revealed several significant differences in the distribution of cells, both intra- and inter-age groups, namely decreased values of T-CD3, T-CD8 and NK cells, and elevated values for T-CD4, T-CD4/T-CD8 ratio and B cells. The percentages of unstimulated neutrophils that show basal oxidative activity and the intensity of this activity had an increasing tendency age-related. The percentage of N-Formyl-Methionyl-Leucyl-Phenylalanine stimulated neutrophils clearly decreases with age, and is associated with an increasing intensity of oxidative activity. Our data also have shown an increased percentage of oxidative neutrophils after phorbol 12-myristate 13-acetate stimulation and an elevated oxidative activity with age.
CONCLUSION
Overall healthy ageing is governed by some immune-related deregulations that account for immune exhaustion due to numerous developed immune processes during a life-time and the age-related diseases.
Topics: Acetates; Aged; Aged, 80 and over; Healthy Aging; Humans; Myristates; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate
PubMed: 36247200
DOI: 10.2147/CIA.S375926 -
PloS One 2022Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and...
Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and inflammation within the gut-liver axis. Studying the hepatic immune response is of high importance as the liver is primarily exposed to gut-derived pathogen-associated molecular patterns. The aim of the present study was to assess the effects of chicken cathelicidin-2 alone or combined with lipoteichoic acid (LTA) or phorbol myristate acetate (PMA) on cell viability, immune response and redox homeostasis in a primary hepatocyte-non-parenchymal cell co-culture of chicken origin. Both concentrations of cathelicidin-2 decreased the cellular metabolic activity and increased the extracellular lactate dehydrogenase (LDH) activity reflecting reduced membrane integrity. Neither LTA nor PMA affected these parameters, and when combined with LTA, cathelicidin-2 could not influence the LDH activity. Cathelicidin-2 had an increasing effect on the concentration of the proinflammatory CXCLi2 and interferon- (IFN-)γ, and on that of the anti-inflammatory IL-10. Meanwhile, macrophage colony stimulating factor (M-CSF), playing a complex role in inflammation, was diminished by the AMP. LTA elevated IFN-γ and decreased M-CSF levels, while PMA only increased the concentration of M-CSF. Both concentrations of cathelicidin-2 increased the H2O2 release of the cells, but the concentration of malondialdehyde as a lipid peroxidation marker was not affected. Our findings give evidence that cathelicidin-2 can also possess anti-inflammatory effects, reflected by the alleviation of the LTA-triggered IFN-γ elevation, and by reducing the M-CSF production induced by PMA. Based on the present results, cathelicidin-2 plays a substantial role in modulating the hepatic immune response with a multifaceted mode of action. It was found to have dose-dependent effects on metabolic activity, membrane integrity, and reactive oxygen species production, indicating that using it in excessively high concentrations can contribute to cell damage. In conclusion, cathelicidin-2 seems to be a promising candidate for future immunomodulating drug development with an attempt to reduce the application of antibiotics.
Topics: Adenosine Monophosphate; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Chickens; Coculture Techniques; Hepatocytes; Hydrogen Peroxide; Immunity; Inflammation; Interferon-gamma; Interleukin-10; Lactate Dehydrogenases; Liver; Macrophage Colony-Stimulating Factor; Malondialdehyde; Pathogen-Associated Molecular Pattern Molecules; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Cathelicidins
PubMed: 36215285
DOI: 10.1371/journal.pone.0275847 -
ELife Oct 2022Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs...
Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/ knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/ KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2 traffics to the plasma membrane with a time-course matching Ca-dependent secretion, and trafficking is independent of Synaptotagmin-7 (Syt7). However, in the absence of Syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2-driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends on functional pairing with Syt7. Overall, DAG/phorbolester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.
Topics: Animals; Mice; Dense Core Vesicles; Diglycerides; Exocytosis; Phorbol Esters; SNARE Proteins; Synaptotagmins
PubMed: 36214779
DOI: 10.7554/eLife.79433 -
Immunology and Cell Biology Nov 2022For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate,...
For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.
Topics: Humans; Real-Time Polymerase Chain Reaction; THP-1 Cells; Lipopolysaccharides; Macrophages; Monocytes; Tetradecanoylphorbol Acetate; Gene Expression Profiling
PubMed: 36184577
DOI: 10.1111/imcb.12590 -
Neurotoxicology Dec 2022Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level...
Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level of GAP-43 in the striatum of rats that receives rich dopaminergic terminals. Using PC12 cells as dopaminergic neuronal models, we further found that AMPH inhibited GAP-43 and GAP-43 phosphorylation in PC12 cells. The reduced GAP-43 was correlated with neurite injury of PC12 cells. The PKCβ1, an upstream molecule of GAP-43, was also inhibited by AMPH. Phorbol 12-myristate 13-acetate (PMA) as a specific activator of PKC increased levels of PKCβ1 and GAP-43, and efficiently prevented neurite degeneration of PC12 cells induced by AMPH. On the other side, enzastuarin, an inhibitor of PKC, decreased levels of PKCβ1 and GAP-43, and caused neurite injury of PC12 cells. Together, our results suggest that AMPH induces neurite injury in PC12 cells through inhibiting PKCβ1/GAP-43 pathway.
Topics: Animals; Rats; Amphetamine; PC12 Cells; Neurites; GAP-43 Protein; Tetradecanoylphorbol Acetate; Dopamine
PubMed: 36150536
DOI: 10.1016/j.neuro.2022.09.004 -
Computational and Structural... 2022Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase...
Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound , as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.
PubMed: 36147668
DOI: 10.1016/j.csbj.2022.08.046 -
Cells Sep 2022This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs.... (Review)
Review
This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, β and δ contribute to ROS production while PKC βII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1β release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.
Topics: Actins; Extracellular Traps; NF-kappa B; Neutrophils; Pancreatic Elastase; Reactive Oxygen Species; Serine Proteases; Tetradecanoylphorbol Acetate
PubMed: 36139464
DOI: 10.3390/cells11182889 -
Biomolecules Sep 2022Bilirubin (BR) is a tetrapyrrolic compound stemming from heme catabolism with diverse physiological functions. It can be oxidized by HO to form several degradation...
Bilirubin (BR) is a tetrapyrrolic compound stemming from heme catabolism with diverse physiological functions. It can be oxidized by HO to form several degradation products, some of which have been detected in vivo and may contribute to the pathogenesis of certain diseases. However, the oxidative degradation of BR is complex and the conditions that BR degradation occurs pathophysiologically remain obscure. Neutrophils are known to generate large amounts of reactive oxygen species, including HO, upon activation and they are mobilized to inflammatory sites; therefore, we hypothesized that activated neutrophils could cause BR degradation, which could occur at inflammatory sites. In the present study, we investigated BR degradation by HO and identified hematinic acid (BHP1) and a new product BHP2, whose structure was characterized as 2,5-diformyl-4-methyl-1-pyrrole-3-propanoic acid. An LC-MS/MS method for the quantitation of the two compounds was then established. Using the LC-MS/MS method, we observed the concentration-dependent formation of BHP1 and BHP2 in mouse neutrophils incubated with 10 and 30 μM of BR with the yields being 16 ± 3.2 and 31 ± 5.9 pmol/10 cells for BHP1, and 25 ± 4.4 and 71 ± 26 pmol/10 cells for BHP2, respectively. After adding phorbol 12-myristate 13-acetate, a neutrophil agonist, to 30 μM of BR-treated cells, the BHP1 yield increased to 43 ± 6.6 pmol/10 cells, whereas the BHP2 one decreased to 47 ± 9.2 pmol/10 cells. The two products were also detected in hemorrhagic skins of mice with dermal inflammation and hemorrhage at levels of 4.5 ± 1.9 and 0.18 ± 0.10 nmol/g tissue, respectively, which were significantly higher than those in the non-hemorrhagic skins. BHP2 was neurotoxic starting at 0.10 μM but BHP1 was not, as assessed using as the animal model. Neutrophil-mediated BR degradation may be a universally pathophysiological process in inflammation and can be particularly important under pathological conditions concerning hemorrhage.
Topics: Acetates; Animals; Bilirubin; Chromatography, Liquid; Heme; Hydrogen Peroxide; Inflammation; Mice; Myristates; Neutrophils; Propionates; Reactive Oxygen Species; Tandem Mass Spectrometry; Tetradecanoylphorbol Acetate
PubMed: 36139076
DOI: 10.3390/biom12091237 -
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