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Current Research in Physiology 2023High affinity methylaminoisobutyric acid(MeAIB)/glutamine(Gln) transport activity regulated by neuronal firing occurs at the plasma membrane in mature rat hippocampal...
High affinity methylaminoisobutyric acid(MeAIB)/glutamine(Gln) transport activity regulated by neuronal firing occurs at the plasma membrane in mature rat hippocampal neuron-enriched cultures. Spontaneous Ca-regulated transport activity was similarly inhibited by riluzole, a benzothiazole anticonvulsant agent, and by novel naphthalenyl substituted aminothiazole derivatives such as SKA-378. Here, we report that spontaneous transport activity is stimulated by 4-aminopyridine (4-AP) and that phorbol-myristate acetate (PMA) increases high K stimulated transport activity that is inhibited by staurosporine. 4-AP-stimulated spontaneous and PMA-stimulated high K-induced transport is not present at 7 days (DIV) and is maximal by DIV∼21. The relative affinity for MeAIB is similar for spontaneous and high K-stimulated transport (K ∼ 50 μM) suggesting that a single transporter is involved. While riluzole and SKA-378 inhibit spontaneous transport with equal potency (IC ∼ 1 μM), they exhibit decreased (∼3-5 X) potency for 4-AP-stimulated spontaneous transport. Interestingly, high K-stimulated MeAIB transport displays lower and differential sensitivity to the two compounds. SKA-378-related halogenated derivatives of SKA-75 (SKA-219, SKA-377 and SKA-375) preferentially inhibit high K-induced expression of MeAIB transport activity at the plasma membrane (IC < 25 μM), compared to SKA-75 and riluzole (IC > 100 μM). Ca-dependent spontaneous and high K-stimulated MeAIB transport activity is blocked by ω-conotoxin MVIIC, ω-agatoxin IVA, ω-agatoxin TK (IC ∼ 500 nM) or cadmium ion (IC ∼ 20 μM) demonstrating that P/Q-type Ca channels that are required for activity-regulated presynaptic vesicular glutamate (Glu) release are also required for high-affinity MeAIB transport expression at the plasma membrane. We suggest that neural activity driven and Ca dependent trafficking of the high affinity MeAIB transporter to the plasma membrane is a unique target to understand mechanisms of Glu/Gln recycling in synapses and acute neuroprotection against excitotoxic presynaptic Glu induced neural injury.
PubMed: 38107787
DOI: 10.1016/j.crphys.2023.100109 -
Biological & Pharmaceutical Bulletin Jan 2024Autophagy and M1 macrophage polarization play important roles in the regulation of inflammation in atopic dermatitis (AD). Dictamnine is one of the main ingredients in...
Autophagy and M1 macrophage polarization play important roles in the regulation of inflammation in atopic dermatitis (AD). Dictamnine is one of the main ingredients in Cortex Dictamni, a widely used traditional Chinese medicine for the treatment of dermatitis. In the present study, we investigated the anti-inflammatory effects of dictamnine on AD like skin lesions and M1 macrophage polarization. A 2,4-dinitrofluorobenzene (DNFB) triggered AD like skin lesions models in mice was established to identify the ameliorative effects of dictamnine on AD in vivo. In addition, an M1 macrophage polarization model was co-stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) using phorbol myristate acetate (PMA) differentiated THP-1 cells, to investigate the effect of dictamnine on promoting autophagy and inhibiting inflammatory factor release. Dictamnine suppressed DNFB-induced skin inflammation by inhibiting M1 macrophage polarization, up-regulating the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3) expression, and promoting macrophage autophagy at inflammatory sites. Dictamnine also could reduce the release of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8), and down-regulate the mRNA expression of these genes in LPS-IFN-γ triggered M1 polarized macrophages. Dictamnine ameliorates AD like skin lesions by inhibiting M1 macrophage polarization and promoting autophagy. Hence, dictamnine is expected to be a potential therapeutic candidate for AD.
Topics: Mice; Animals; Dermatitis, Atopic; Dinitrofluorobenzene; Lipopolysaccharides; Inflammation; Macrophages; Autophagy; Interferon-gamma; Quinolines
PubMed: 38092386
DOI: 10.1248/bpb.b23-00436 -
Iranian Journal of Allergy, Asthma, and... Oct 2023Programmed death ligand‑1 (PD‑L1) is a pivotal inhibitory checkpoint ligand known to induce T-cell exhaustion via interaction with the programmed death‑1 (PD‑1)...
Programmed death ligand‑1 (PD‑L1) is a pivotal inhibitory checkpoint ligand known to induce T-cell exhaustion via interaction with the programmed death‑1 (PD‑1) receptor. Beyond this, PD-L1's intrinsic signaling pathways within cancer cells warrant further exploration. This study aims to elucidate the effect of PD-L1 stimulation on the proliferation, survival, and apoptosis of acute myeloid leukemia (AML) cell lines. Two human AML cell lines, HL-60 and THP-1 were cultured and treated with phorbol 12-myristate 13-acetate (PMA) to induce PD-L1overexpression. Post-treatment PD-L1 expression was confirmed via flow cytometry. Subsequently, cell surface PD-L1 was stimulated using a recombinant PD-1, 24 hours post-PMA treatment. The expression alterations in pivotal genes including BCL2, MKI67, BAX, and CASP3 were monitored using quantitative real-time polymerase chain reaction 24 and 48 hours post-treatment. Additionally, annexin-V through flow cytometry. Findings reveal that PD-L1 stimulation augments AML cell proliferation and survival by enhancing MKI67 and BCL2 expressions while concurrently inhibiting cell apoptosis due to decreased BAX and CASP3 expression following PD-L1 stimulation. Notably, stimulated cells expressed exhibited reduced annexin-V compared to control cells. This study underscores that PD-L1 stimulation fosters AML cell proliferation and survival while impeding cell apoptosis. The results hold potential implications for targeting PD-L1 in AML treatment strategies.
Topics: Humans; Programmed Cell Death 1 Receptor; bcl-2-Associated X Protein; B7-H1 Antigen; Ki-67 Antigen; Ligands; Caspase 3; Leukemia, Myeloid, Acute; Apoptosis; Cell Proliferation; Annexins; Proto-Oncogene Proteins c-bcl-2
PubMed: 38085150
DOI: 10.18502/ijaai.v22i5.13998 -
Journal of Pharmacological Sciences Jan 2024The DNA recognition peptide compounds pyrrole-imidazole (PI) polyamides bind to the minor groove and can block the binding of transcription factors to target sequences....
PURPOSE
The DNA recognition peptide compounds pyrrole-imidazole (PI) polyamides bind to the minor groove and can block the binding of transcription factors to target sequences. To develop more PI polyamides as potential treatments for fibrotic diseases, including chronic renal failure, we developed multifunctional PI polyamides that increase hepatocyte growth factor (HGF) and decrease transforming growth factor (TGF)-β1.
METHODS
We designed seven PI polyamides (HGF-1 to HGF-7) that bind to the chicken ovalbumin upstream promoter transcription factor-1 (COUP-TF1) binding site of the HGF promoter sequence. We selected PI polyamides that increase HGF and suppress TGF-β1 in human dermal fibroblasts (HDFs).
FINDINGS
Gel shift assays showed that HGF-2 and HGF-4 bound the appropriate dsDNAs. HGF-2 and HGF-4 significantly inhibited the TGF-β1 mRNA expression in HDFs stimulated by phorbol 12-myristate 13-acetate. HGF-2 and HGF-4 significantly inhibited the TGF-β1 protein expression in HDFs with siRNA targeting HGF, indicating that HGF-2 and HGF-4 directly inhibited the expression of TGF-β1.
CONCLUSION
The designed and synthetic HGF PI polyamides targeting the HGF promoter, which increased the expression of HGF and suppressed the expression of TGF-β, will be a potential practical medicine for fibrotic diseases, including progressive renal diseases.
Topics: Humans; Transforming Growth Factor beta1; Nylons; Hepatocyte Growth Factor; Transforming Growth Factor beta; Pyrroles; Imidazoles
PubMed: 38081679
DOI: 10.1016/j.jphs.2023.11.001 -
Infectious Medicine Sep 2023Given that epidemiological evidence suggests a potential protective role for Bacille-Calmette-Guerin against COVID-19, we aimed to explore whether pre-exposure of human...
BACKGROUND
Given that epidemiological evidence suggests a potential protective role for Bacille-Calmette-Guerin against COVID-19, we aimed to explore whether pre-exposure of human monocyte-derived macrophages and dendritic cells to BCG could modulate their response to SARS-CoV-2 S-glycoprotein.
METHODS
Dual THP-1 cells containing 2 reporter plasmids for transcription factors NF-κB, and IRF were differentiated into macrophages over 3 days using phorbol 12-myristate 13-acetate, or into dendritic cells over 6 days using commercial monocyte-dencritic cell differentiation media and matured with recombinant tumor necrosis factor-α. Cells were exposed to BCG for 24 h and then stimulated with SARS-CoV-2 S-glycoprotein for 24 hours.
RESULTS
Pre-exposure of human macrophages and DCs to BCG increased IRF and NF-kb activation in response to the SARS-CoV-2 S-glycoprotein.
CONCLUSIONS
Our results showed that pre-exposure of both types of cells to BCG exhibited an increase in inflammatory transcription factors upon stimulation with S-glycoprotein. BCG-induced trained immunity may be an important tool for reducing susceptibility to SARS-CoV-2 infection and severity of COVID-19. Our findings help in the design of future BCG-based therapeutic approaches in the treatment of diseases caused by viral infections.
PubMed: 38073885
DOI: 10.1016/j.imj.2023.08.004 -
International Journal of Molecular... Nov 2023The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with...
The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.
Topics: Phosphorylation; Calcium; Norepinephrine; Phorbol Esters; Receptors, Adrenergic
PubMed: 38069285
DOI: 10.3390/ijms242316963 -
Free Radical Biology & Medicine Jan 2024Recent investigations have proposed a potential causal association between the occurrence of ferroptosis, nuclear factor kappa B (NF-κB) and ubiquitin-specific protease...
BACKGROUND
Recent investigations have proposed a potential causal association between the occurrence of ferroptosis, nuclear factor kappa B (NF-κB) and ubiquitin-specific protease 24 (USP24). Nevertheless, the mechanism of USP24 and NF-κB regulation of ferroptosis in the context of diabetic cardiomyopathy (DCM) remain unclear.
METHODS
In this study, a high-fat diet and a streptozotocin-induced mouse DCM model were established, and high glucose and palmitic acid treatment of H9c2 cells and neonatal mouse primary cardiomyocytes (NMPCs) was used as an in vitro DCM models. Utilizing both the in vivo and in vitro DCM models, we assessed of USP24, NF-κB, and ferroptosis levels, and explored the relationship among them.
RESULTS
In in vivo and in vitro DCM models, increased expression of USP24, NF-κB, phosphorylated NF-κB (p-NF-κB) and fatty acid-CoA ligase 4 (FACL4) were detected, along with accumulated iron, as well as reduced ferritin heavy chain 1 (FTH1), solute carrier family 7 member 11 (SLC7A11) and antioxidant capacity. Knockdown of USP24 resulted in a reduction of NF-κB levels, while knockdown of NF-κB did not lead to a decrease in USP24 expression. Moreover, in H9c2 cells, knockdown of USP24 and NF-κB separately resulted in reduced levels of FACL4, increased levels of SLC7A11 and FTH1, as well as improved antioxidant capacity and cell viability. In shUSP24 knockdown H9c2 cells, administration of phorbol 12-myristate 13-acetate (PMA) activated NF-κB, subsequently reversing the previously observed effect caused by USP24 knockdown.
CONCLUSIONS
These findings show that USP24 upregulates NF-κB to promote ferroptosis in DCM.
Topics: Animals; Mice; Antioxidants; Diabetes Mellitus; Diabetic Cardiomyopathies; Ferroptosis; NF-kappa B; Up-Regulation
PubMed: 38056575
DOI: 10.1016/j.freeradbiomed.2023.11.032 -
Frontiers in Immunology 2023Patients with primary adrenal insufficiency (PAI) suffer from increased risk of infection, adrenal crises and have a higher mortality rate. Such dismal outcomes have...
INTRODUCTION
Patients with primary adrenal insufficiency (PAI) suffer from increased risk of infection, adrenal crises and have a higher mortality rate. Such dismal outcomes have been inferred to immune cell dysregulation because of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid (GC) deficiency.
METHODS
This cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 after bilateral adrenalectomy due to Cushing's syndrome (BADx), 21 with Addison's disease (AD) and 52 healthy controls. All patients with PAI were on a stable GC replacement regimen with a median dose of 25 mg hydrocortisone per day. Peripheral blood mononuclear cells were isolated from heparinized blood samples. Immune cell subsets were analyzed using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate and ionomycin. Natural killer (NK-) cell cytotoxicity and clock gene expression were investigated.
RESULTS
The percentage of T helper cell subsets was downregulated in AD patients (Th1 p = 0.0024, Th2 p = 0.0157, Th17 p < 0.0001) compared to controls. Cytotoxic T cell subsets were reduced in AD (Tc1 p = 0.0075, Tc2 p = 0.0154) and CAH patients (Tc1 p = 0.0055, Tc2 p = 0.0012) compared to controls. NKCC was reduced in all subsets of PAI patients, with smallest changes in CAH. Degranulation marker CD107a expression was upregulated in BADx and AD, not in CAH patients compared to controls (BADx p < 0.0001; AD p = 0.0002). In contrast to NK cell activating receptors, NK cell inhibiting receptor CD94 was upregulated in BADx and AD, but not in CAH patients (p < 0.0001). Although modulation in clock gene expression could be confirmed in our patient subgroups, major interindividual-intergroup dissimilarities were not detected.
DISCUSSION
In patients with different etiologies of PAI, distinct differences in T and NK cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight unsuspected differences in immune cell composition and function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment.
Topics: Humans; Addison Disease; Cross-Sectional Studies; Leukocytes, Mononuclear; Cushing Syndrome; Glucocorticoids; Hydrocortisone; Adrenal Hyperplasia, Congenital; Adrenal Insufficiency
PubMed: 38045693
DOI: 10.3389/fimmu.2023.1275828 -
Journal of Microbiology and... Feb 2024Basophils and mast cells are specialized effector cells in allergic reactions. (abalone), is valuable seafood. Abalone male viscera, which has a brownish color and has...
Basophils and mast cells are specialized effector cells in allergic reactions. (abalone), is valuable seafood. Abalone male viscera, which has a brownish color and has not been previously reported to show anti-allergic activities, was extracted with acetone. Six different acetone/hexane fractions (0, 10, 20, 30, 40, and 100%) were obtained using a silica column via β-hexosaminidase release inhibitory activity-guided selection in phorbol myristate acetate and a calcium ionophore, A23187 (PMACI)-induced human basophils, KU812F cells. The 40% acetone/hexane fraction (A40) exhibited the strongest inhibition of PMACI-induced-β-hexosaminidase release. This fraction dose-dependently inhibited reactive oxygen species (ROS) production and calcium mobilization without cytotoxicity. Western blot analysis revealed that A40 down-regulated PMACI-induced MAPK (ERK 1/2, p-38, and JNK) phosphorylation, and the NF-κB translocation from the cytosol to membrane. Moreover, A40 inhibited PMACI-induced interleukin (IL)-1β, IL-6, and IL-8 production. Anti-allergic activities of A40 were confirmed based on inhibitory effects on IL-4 and tumor necrosis factor alpha (TNF-α) production in compound (com) 48/80-induced rat basophilic leukemia (RBL)-2H3 cells. A40 inhibited β-hexosaminidase release and cytokine production such as IL-4 and TNF-α produced by com 48/80-stimulated RBL-2H3 cells. Furthermore, it's fraction attenuated the IgE/DNP-induced passive cutaneous anaphylaxis (PCA) reaction in the ears of BALB/c mice. Our results suggest that abalone contains the active fraction, A40 is a potent therapeutic and functional material to treat allergic diseases.
Topics: Rats; Mice; Male; Humans; Animals; Anaphylaxis; Tumor Necrosis Factor-alpha; Basophils; Hexanes; Immunoglobulin E; Acetone; Interleukin-4; Viscera; Anti-Allergic Agents; p-Methoxy-N-methylphenethylamine; beta-N-Acetylhexosaminidases; Cytokines
PubMed: 38037338
DOI: 10.4014/jmb.2310.10015 -
Pharmaceutics Nov 2023This study explores the potential of a natural composite formulation known as ED, consisting of ( family: Lessoniaceae) and Linne (, family: Asteraceae), in...
Effect of and Complex Extract on Phorbol 12-Myristate 13-Acetate (PMA)-Stimulated Inflammatory Response in Human Pulmonary Epithelial Cells and Particulate Matter (PM)-Induced Pulmonary Inflammation in Mice.
This study explores the potential of a natural composite formulation known as ED, consisting of ( family: Lessoniaceae) and Linne (, family: Asteraceae), in alleviating lung inflammation induced by fine particulate matter (PM). Initial assessments confirmed that neither ED nor one of its components, dieckol, exhibited cytotoxic effects on A549 cells. Subsequently, the impact of ED and dieckol on gene expression in A549 cells stimulated by phorbol 12-myristate 13-acetate (PMA) was investigated, revealing promising results that demonstrated a dose-dependent inhibition of gene expression. The study also delves into the underlying mechanisms, demonstrating that ED and dieckol effectively suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38, which are known to be involved in the regulation of gene expression. In in vivo experiments using a PM-induced pulmonary inflammation mouse model, the research findings showed that ED mitigated cellular accumulation in the airways, leading to a significant reduction in the total cell count in bronchoalveolar lavage fluid (BALF). Moreover, ED exhibited protective effects against PM-induced pulmonary damage, characterized by reduced inflammatory cell infiltration and decreased mucus secretion in pulmonary tissues. Additionally, ED's anti-inflammatory properties were evident in its ability to decrease the levels of key inflammatory cytokines, and , both in the serum and lung tissue of the PM-induced pulmonary inflammation mouse model. These findings suggest the potential of ED as a therapeutic agent for inflammatory respiratory diseases.
PubMed: 38004599
DOI: 10.3390/pharmaceutics15112621