Search Results (11)

Early cellular alterations in abdominal aortic aneurysm (AAA) are scarcely investigated. Aortic remodeling inflammation-related suggested the CXCR2/CXCL1/IL-8 axis as a therapeutic target. This study investigates CXCR1/CXCR2 antagonism in primary human aortic endothelial (HAOEC) and smooth muscle cells (HAOSMC) conditioned with IL-8 or serum from patients with AAA (sPT). Ladarixin (10 μM Lad or 25 μM) served as an inhibitor. Readouts included RT-qPCR for CXCL1, CXCL8, CXCR2, MMP9, NFKB1, and VEGF-A; zymography for MMP9 activity confocal microscopy for F-actin and mitochondria; NADPH/NADH diaphorase histochemistry for redox activity; and ATP assay. In HAOEC, IL-8 downregulated CXCR2, increased MMP9 activity, and induced cytoskeletal and mitochondria disorganization without altering NADH/NADPH diaphorases but increasing ATP release. At concentration of 10 μM Lad rescued cell organization and gene expression. sPT upregulated CXCL8, CXCR2, and MMP9, decreased NADH/NADPH diaphorases, and altered cytoskeleton and mitochondria organization in HAOEC. At concentration of 10 μM Lad (partially) and 25 μM Lad reverted gene upregulation and mitochondria distribution; both doses increased diaphorase and released ATP. HAOSMC were scantily susceptible to IL-8 and weakly responsive to sPT, slightly upregulating CXCR2 and VEGF-A but increasing proMMP9 gelatinolysis. Ladarixin recovered proMMP9 activity and modulated CXCL1. AAA-like vascular cell alterations involve multiple inflammatory factors and are modulable by inhibition of IL-8 receptors. The results underline careful dose calibration. Full article

Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3–5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X–Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues. Full article

Our recent investigations indicated that isoforms of ferredoxin (Fd) and ferredoxin NADP⁺ reductase (FNR) play essential roles for the reductive steps of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in peppermint glandular trichomes (GTs). Based on an analysis of several transcriptome data sets, we demonstrated the presence of transcripts for a leaf-type FNR (L-FNR), a leaf-type Fd (Fd I), a root-type FNR (R-FNR), and two root-type Fds (Fd II and Fd III) in several members of the mint family (Lamiaceae). The present study reports on the biochemical characterization of all Fd and FNR isoforms of peppermint (Mentha × piperita L.). The redox potentials of Fd and FNR isoforms were determined using photoreduction methods. Based on a diaphorase assay, peppermint R-FNR had a substantially higher specificity constant (k_cat/K_m) for NADPH than L-FNR. Similar results were obtained with ferricyanide as an electron acceptor. When assayed for NADPH–cytochrome c reductase activity, the specificity constant with the Fd II and Fd III isoforms (when compared to Fd I) was slightly higher for L-FNR and substantially higher for R-FNR. Based on real-time quantitative PCR assays with samples representing various peppermint organs and cell types, the Fd II gene was expressed very highly in metabolically active GTs (but also present at lower levels in roots), whereas Fd III was expressed at low levels in both roots and GTs. Our data provide evidence that high transcript levels of Fd II, and not differences in the biochemical properties of the encoded enzyme when compared to those of Fd III, are likely to support the formation of copious amounts of monoterpene via the MEP pathway in peppermint GTs. This work has laid the foundation for follow-up studies to further investigate the roles of a unique R-FNR–Fd II pair in non-photosynthetic GTs of the Lamiaceae. Full article

NADPH:thioredoxin reductase (TrxR) is considered a potential target for anticancer agents. Several nitroheterocyclic sulfones, such as Stattic and Tri-1, irreversibly inhibit TrxR, which presumably accounts for their antitumor activity. However, it is necessary to distinguish the roles of enzymatic redox cycling, an inherent property of nitroaromatics (ArNO₂), and the inhibition of TrxR in their cytotoxicity. In this study, we calculated the previously unavailable values of single-electron reduction potentials of known inhibitors of TrxR (Stattic, Tri-1, and 1-chloro-2,4-dinitrobenzene (CDNB)) and inhibitors identified (nitrofuran NSC697923 and nitrobenzene BTB06584). These calculations were according to the rates of their enzymatic single-electron reduction (PMID: 34098820). This enabled us to compare their cytotoxicity with that of model redox cycling ArNO₂. In MH22a and HCT-116 cells, Tri-1, Stattic, CDNB, and NSC697023 possessed at least 10-fold greater cytotoxicity than can be expected from their redox cycling activity. This may be related to TrxR inhibition. The absence of enhanced cytotoxicity in BTB06548 may be attributed to its instability. Another known inhibitor of TrxR, tetryl, also did not possess enhanced cytotoxicity, probably because of its detoxification by DT-diaphorase (NQO1). Apart from the reactions with NQO1, the additional mechanisms influencing the cytotoxicity of the examined inhibitors of TrxR are their reactions with cytochromes P-450. Furthermore, some inhibitors, such as Stattic and NSC697923, may also inhibit glutathione reductase. We suggest that these data may be instrumental in the search for TrxR inhibitors with enhanced cytotoxic/anticancer activity. Full article

The birch tree-derived pentacyclic lupine type-triterpenoid Betulinic acid has demonstrated a variety of biological activities BetA is known for its harmlessness on normal healthy cells. However, recent investigations have indicated that BetA can cause cellular changes in mouse normal embryonic fibroblasts even with a minimal concentration. This report cautioned the use of BetA at the clinical level, which encouraged us to examine whether BetA could produce any key effect on normal healthy cells of any organs in mice. The present study extended its investigation to evaluate whether BetA could induce any changes in the renal system and the expression pattern of NADPH-diaphorase an indirect marker of the enzyme nitric oxide synthase in mice. Our results indicated that BetA exposure induced NADPH-d expression in both organs without causing any significant morphological changes. Moreover, NADPH-d activity patterns in the organs of BetA-treated animals tremendously increased (from day 4 until day 12) when compared to controls. The expression of NADPH-d in both the kidney and bladder implies that NADPH-d-mediated nitric oxide signaling could be a mechanism involved in BetA-induced nephroprotection. These outcomes are of direct clinical importance and could pay the way for the improvement of BetA as an important pharmaceutical product. Full article

Excitotoxicity is a form of neuronal death characterized by the sustained activation of N-methyl-D-aspartate receptors (NMDARs) triggered by the excitatory neurotransmitter glutamate. NADPH-diaphorase neurons (also known as nNOS (+) neurons) are a subpopulation of aspiny interneurons, largely spared following excitotoxic challenges. Unlike nNOS (−) cells, nNOS (+) neurons fail to generate reactive oxygen species in response to NMDAR activation, a critical divergent step in the excitotoxic cascade. However, additional mechanisms underlying the reduced vulnerability of nNOS (+) neurons to NMDAR-driven neuronal death have not been explored. Using functional, genetic, and molecular analysis in striatal cultures, we indicate that nNOS (+) neurons possess distinct NMDAR properties. These specific features are primarily driven by the peculiar redox milieu of this subpopulation. In addition, we found that nNOS (+) neurons exposed to a pharmacological maneuver set to mimic chronic excitotoxicity alter their responses to NMDAR-mediated challenges. These findings suggest the presence of mechanisms providing long-term dynamic regulation of NMDARs that can have critical implications in neurotoxic settings. Full article

FNR and ferredoxin constitute a redox cascade, which provides reducing power in the plastid of malaria parasites. Recently, mutation of ferredoxin (D97Y) was reported to be strongly related to the parasite’s resistance to the front-line antimalarial drug artemisinin. In order to gain insight into the mechanism for the resistance, we studied the effect of dihydroartemisinin (DHA), the active compound of artemisinin, on the redox cascade of NADPH/FNR/ferredoxin in in vitro reconstituted systems. DHA partially inhibited the diaphorase activity of FNR by decreasing the affinity of FNR for NADPH. The activity of the electron transfer from FNR to wild-type or D97Y mutant ferredoxin was not significantly affected by DHA. An in silico docking analysis indicated possible binding of DHA molecule in the binding cavity of 2′5′ADP, a competitive inhibitor for NADPH, on FNR. We previously showed that the D97Y mutant of ferredoxin binds to FNR more strongly than wild-type ferredoxin, and ferredoxin and FNR are generally known to be involved in the oxidative stress response. Thus, these results suggest that ferredoxin is not a direct target of artemisinin, but its mutation may be involved in the protective response against the oxidative stress caused by artemisinin. Full article

Thymoquinone (TQ) is the main biologically active constituent of Nigella sativa. Many studies have confirmed its anticancer actions. Herein, we investigated the different anticancer activities of, and considered resistance mechanisms to, TQ. MTT and clonogenic data showed TQ’s ability to suppress breast MDA-MB-468 and T-47D proliferation at lower concentrations compared to other cancer and non-transformed cell lines tested (GI₅₀ values ≤ 1.5 µM). Flow-cytometric analyses revealed that TQ consistently induced MDA-MB-468 and T-47D cell-cycle perturbation, specifically inducing pre-G1 populations. In comparison, less sensitive breast MCF-7 and colon HCT-116 cells exhibited only transient increases in pre-G1 events. Annexin V/PI staining confirmed apoptosis induction in MDA-MB-468 and HCT-116 cells, which was continuous in the former and transient in the latter. Experiments revealed the role of reactive oxygen species (ROS) generation and aneuploidy induction in MDA-MB-468 cells within the first 24 h of treatment. The ROS-scavenger NAD(P)H dehydrogenase (quinone 1) (NQO1; DT-diaphorase) and glutathione (GSH) were implicated in resistance to TQ. Indeed, western blot analyses showed that NQO1 is expressed in all cell lines in this study, except those most sensitive to TQ-MDA-MB-468 and T-47D. Moreover, TQ treatment increased NQO1 expression in HCT-116 in a concentration-dependent fashion. Measurement of GSH activity in MDA-MB-468 and HCT-116 cells found that GSH is similarly active in both cell lines. Furthermore, GSH depletion rendered these cells more sensitive to TQ’s antiproliferative actions. Therefore, to bypass putative inactivation of the TQ semiquinone metabolite, the benzylamine analogue was designed and synthesised following modification of TQ’s carbon-3 atom. However, the structural modification negatively impacted potency against MDA-MB-468 cells. In conclusion, we disclose the following: (i) The anticancer activity of TQ may be a consequence of ROS generation and aneuploidy; (ii) Early GSH depletion could substantially enhance TQ’s anticancer activity; (iii) Benzylamine substitution at TQ’s carbon-3 failed to enhance anticancer activity. Full article

Lichens are poikilohydrous symbiotic associations between a fungus, photosynthetic partners, and bacteria. They are tolerant to repeated desiccation/rehydration cycles and adapted to anhydrobiosis. Nitric oxide (NO) is a keystone for stress tolerance of lichens; during lichen rehydration, NO limits free radicals and lipid peroxidation but no data on the mechanisms of its synthesis exist. The aim of this work is to characterize the synthesis of NO in the lichen Ramalina farinacea using inhibitors of nitrate reductase (NR) and nitric oxide synthase (NOS), tungstate, and NG-nitro-L-arginine methyl ester (L-NAME), respectively. Tungstate suppressed the NO level in the lichen and caused an increase in malondialdehyde during rehydration in the hyphae of cortex and in phycobionts, suggesting that a plant-like NR is involved in the NO production. Specific activity of NR in R. farinacea was 91 μU/mg protein, a level comparable to those in the bryophyte Physcomitrella patens and Arabidopsis thaliana. L-NAME treatment did not suppress the NO level in the lichens. On the other hand, NADPH-diaphorase activity cytochemistry showed a possible presence of a NOS-like activity in the microalgae where it is associated with cytoplasmatic vesicles. These data provide initial evidence that NO synthesis in R. farinacea involves NR. Full article

Cytochrome b5 reductase 3 (CYB5R3) is a membrane-bound NADH-dependent redox enzyme anchored to the mitochondrial outer membrane, endoplasmic reticulum, and plasma membrane. Recessive hereditary methaemoglobinaemia (RHM) type II is caused by CYB5R3 deficiency and is an incurable disease characterized by severe encephalopathy with mental retardation, microcephaly, generalized dystonia, and movement disorders. Currently, the etiology of type II RHM is poorly understood and there is no treatment for encephalopathy associated with this disease. Defective CYB5R3 leads to defects in the elongation and desaturation of fatty acids and cholesterol biosynthesis, which are conventionally linked with neurological disorders of type II RHM. Nevertheless, this abnormal lipid metabolism cannot explain all manifestations observed in patients. Current molecular and cellular studies indicate that CYB5R3 deficiency has pleiotropic tissue effects. Its localization in lipid rafts of neurons indicates its role in interneuronal contacts and its presence in caveolae of the vascular endothelial membrane suggests a role in the modulation of nitric oxide diffusion. Its role in aerobic metabolism and oxidative stress in fibroblasts, neurons, and cardiomyocytes has been reported to be due to its ability to modulate the intracellular ratio of NAD⁺/NADH. Based on the new molecular and cellular functions discovered for CYB5R3 linked to the plasma membrane and mitochondria, the conventional conception that the cause of type II RHM is a lipid metabolism disorder should be revised. We hypothesized that neurological symptoms of the disease could be caused by disorders in the synapse, aerobic metabolism, and/or vascular homeostasis rather than in disturbances of lipid metabolism. Full article

Nitric oxide (NO) is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS) from the conversion of L-arginine to citrulline. Supplementation of L-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome), a mitochondrial disease caused by the m.3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. In this study we found that osteosarcoma derived cybrid cells with high levels of m.3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM). Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m.3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of L-arginine effect to determine the appropriate clinical use of this drug therapy. Full article