Oxidase Complex Research Articles

Inhibition and evasion of neutrophil microbicidal responses by Legionella longbeachae.

Legionella longbeachae is commonly found in soil environments where it interacts with a wide variety of protist hosts and microbial competitors. Upon transmission to humans, L. longbeachae invades and replicates within alveolar macrophages, leading to the manifestation of pneumonia. In addition to alveolar macrophages, neutrophils are abundant immune cells acting as the first line of defense against invading pathogens. While most intracellular bacterial species are killed and degraded by neutrophils, we show that L. longbeachae evades degradation. The pathogen impairs the major neutrophils' microbicidal processes, including the fusion of microbicidal granules to the pathogen-containing vacuole. By inhibiting of assembly of the phagocyte NADPH oxidase complex, the pathogen blocks neutrophils from generating microbicide reactive oxygen species. Overall, L. longbeachae employs unique virulence strategies to evade the major microbicidal processes of neutrophils.

FC05 Circulating monocytes in psoriasis patients treated with apremilast reveal potential cardioprotective effects

Abstract Psoriasis is a systemic inflammatory condition characterized by inflamed plaques on the skin that is associated with an increased risk of cardiovascular disease (CVD) including myocardial infarction, hypercholesterolaemia, and hypertension. It is hypothesized that the systemic inflammation associated with psoriasis contributes to the development of CVD; however, the precise mechanism(s) and the impact of systemic therapy remain unknown. We previously demonstrated that circulating monocytes from individuals with psoriasis exhibit a hyperactivated adhesive transcriptional gene expression endotype. Additionally, patients with elevated monocyte doublets at baseline had normalization of these levels following treatment with apremilast. This suggests that aberrant adhesive monocytes contribute to CVD in psoriasis and that apremilast may not only be effective at treating cutaneous disease but may also have cardioprotective effects via monocyte modulation. Our goals were to (i) identify the top differentially expressed genes (DEGs) and associated pathways in psoriasis patients circulating CD14+ monocytes following treatment with apremilast through a prospective cohort study, and (ii) determine if psoriasis patients treated with apremilast have a lower relative risk of CVD vs. those treated with topical corticosteroid (TCS) alone through a retrospective cohort analysis. First, we conducted a prospective cohort study of 14 individuals with psoriasis where CD14+ monocytes were negatively selected from whole blood for transcriptome analysis at baseline and 16 weeks post-treatment. Next, a retrospective cohort study using TriNetX was utilized to determine if apremilast treatment in psoriasis is associated with a lower relative risk (RR) for developing CVD within 5 years compared with those receiving TCS. The top DEGs post-treatment included KCNS1, WNT4, and CASS4 (increased) and ID1, ENKUR, and PIM1 (decreased). Key pathways identified were macrophage differentiation, NADPH oxidase complex activation, and lipoprotein clearance. In the retrospective analysis, 9269 individuals in each group (apremilast vs. TCS) were matched for age, sex, body mass index, race, Type 2 diabetes mellitus, nicotine dependence, alcohol-related disorders, chronic kidney disease, aspirin, antilipemic agents and anticoagulation. Comparisons between individuals treated with apremilast vs. TCS revealed that apremilast treatment was associated with a lower RR of new-onset myocardial infarction (0.754; 0.606–0.939), cerebral infarction (0.667; 0.542–0.820), deep vein thrombosis (0.812; 0.662–0.997), and hypertension (0.702; 0.633–0.778). Concordant with the finding that apremilast modified monocytes lipoprotein clearance, apremilast treatment resulted in reduced risk of hypercholesterolaemia (0.671; 0.610–0.738) and elevated low-density lipoprotein (0.628; 0.532–0.742) relative to those receiving TCS alone. In conclusion, apremilast therapy reduces the risk of new-onset CVD, hypertension, and thrombotic events in individuals with psoriasis, which may be mediated by alteration of circulating monocyte reactive oxygen species and lipoprotein clearance.

NCF-1 plays a pivotal role in the survival of adenocarcinoma cells of pancreatic and gastric origins

Reactive oxygen species (ROS) play a pivotal biological role in cells, with ROS function differing depending on cellular conditions and the extracellular environment. Notably, ROS act as cytotoxic factors to eliminate infectious pathogens or promote cell death under cellular stress, while also facilitating cell growth (via ROS-sensing pathways) by modifying gene expression. Among ROS-related genes, neutrophil cytosolic factor-1 (NCF-1; p47phox) was identified as a ROS generator in neutrophils. This product is a subunit of a cytosolic NADPH oxidase complex activated in response to pathogens such as bacteria and viruses. NCF-1 has been examined primarily in terms of ROS-production pathways in macrophages and neutrophils; however, the expression of this protein and its biological role in cancer cells remain unclear. Here, we report expression of NCF-1 in pancreatic and gastric cancers, and demonstrate its biological significance in these tumor cells. Abundant expression of NCF-1 was observed in pancreatic adenocarcinoma (PDAC) lines and in patient tissues, as well as in gastric adenocarcinomas. Accumulation of the protein was also detected in the invasive/metastatic foci of these tumors. Unexpectedly, BxPC-3 underwent apoptotic cell death when transfected with a small interfering RNA (siRNA) specific to NCF-1, whereas the cells treated with a control siRNA proliferated in a time-dependent manner. A similar phenomenon was observed in HSC-58, a poorly differentiated gastric adenocarcinoma line. Consequently, the tumor cells highly expressing NCF-1 obtained coincident accumulation of ROS and reduced glutathione (GSH) with expression of glutathione peroxidase 4 (GPX4), a quencher involved in ferroptosis. Unlike the conventional role of ROS as a representative cytotoxic factor, these findings suggest that NCF-1-mediated ROS generation may be required for expansive growth of PDAC and gastric cancers.

Development of Convenient Immunosensors Employing Single Chain Variable Fragment (scFv)-Displayed Biogenic Magnetic Nanoparticles

Introduction Convenient immunosensors are desired for the early diagnosis of diseases; however, washing operation to remove unreacted sensing elements takes a long time and requires well-trained technicians in conventional immunosensors. Recently, recombinant antibody fragments such as single chain variable fragments (scFvs) have been employed to realize them. Here, we developed two wash-free immunodetection systems combining a scFv-glucose oxidase complex (scFv-GOx) (Fig.1a) and a scFv-displaying magnetic nanoparticle (scFv-MNP) (Fig. 1b). The first one is a chemiluminescent detection system based on the peroxidase activity of MNPs (Fig. 2a). When scFv-MNP and scFv-GOx bind to the target, MNP and GOx become spatially close. Substrate addition is then expected to initiate a target-dependent sequential reaction in which luminol reacts with hydrogen peroxide generated by the oxidation of GOx to produce a chemiluminescent signal. The second one is an electrochemical detection system that integrates magnetic separation (Fig. 2b). Targets are preincubated with scFv-MNP and scFv-GOx, and dropped onto an electrode. Magnetic separation allows efficient electron transfer from GOx to the electrode. Methods To prepare scFv-MNPs, we focused on biogenic MNPs, magnetosomes synthesized by magnetotactic bacterium, Magnetospirillum magneticum. We also integrated an efficient protein coupling module, SpyCatcher (SC)/SpyTag (ST) system, to facilitate the exchange of scFvs depending on the target. First, preparing SC fused Mms13, an endogenously existing protein in the lipid layer of magnetosome, which was utilized for protein display on magnetosomes1, then we isolated SC-displayed MNPs (SC-MNPs) (Fig. 1c) from transformed magnetic bacteria with the vector expressing SC fused Mms 13. Second, to detect C-reactive protein (CRP) with homo pentameric structure, anti-CRP scFv-MNPs were prepared by mixing SC-MNPs and ST-fused anti-CRP scFv (scFv-ST) (Fig. 1d). Then, scFv-MNPs, anti-CRP scFv-GOx, and CRP were mixed and incubated for 30 minutes. Chemiluminescence was measured after addition of glucose and luminol. For the electrochemical detection, the mixture was dropped onto a printed carbon electrode and response current was monitored after the addition of glucose and magnetic separation. Results and discussion CRP concentration-dependent signal increases were observed in the range of 10-250 nM and 10-100 nM, in the chemiluminescent detection and the electrochemical detection, respectively. In the chemiluminescent detection system, a more convenient operation was achieved by simply mixing scFv-MNPs and GOx-scFv, but the signal detection time took about 20 min. In contrast, the electrochemical detection time took around 5 min because the current value increases immediately after glucose is added, but the sensitivity was low under the present detection conditions. Therefore, we are working to optimize the detection conditions and detect CRP in serum with both detection systems. Reference Tanaka et al., Anal. Chem. 2000, 72 (15), 3518−3522. Figure 1

Balanced regulation of ROS production and inflammasome activation in preventing early development of colorectal cancer.

Reactive oxygen species (ROS) production and inflammasome activation are the key components of the innate immune response to microbial infection and sterile insults. ROS are at the intersection of inflammation and immunity during cancer development. Balanced regulation of ROS production and inflammasome activation serves as the central hub of innate immunity, determining whether a cell will survive or undergo cell death. However, the mechanisms underlying this balanced regulation remain unclear. Mitochondria and NADPH oxidases are the two major sources of ROS production. Recently, NCF4, a component of the NADPH oxidase complex that primarily contributes to ROS generation in phagocytes, was reported to balance ROS production and inflammasome activation in macrophages. The phosphorylation and puncta distribution of NCF4 shifts from the membrane-bound NADPH complex to the perinuclear region, promoting ASC speck formation and inflammasome activation, which triggers downstream IL-18-IFN-γ signaling to prevent the progression of colorectal cancer (CRC). Here, we review ROS signaling and inflammasome activation studies in colitis-associated CRC and propose that NCF4 acts as a ROS sensor that balances ROS production and inflammasome activation. In addition, NCF4 is a susceptibility gene for Crohn's disease (CD) and CRC. We discuss the evidence demonstrating NCF4's crucial role in facilitating cell-cell contact between immune cells and intestinal cells, and mediating the paracrine effects of inflammatory cytokines and ROS. This coordination of the signaling network helps create a robust immune microenvironment that effectively prevents epithelial cell mutagenesis and tumorigenesis during the early stage of colitis-associated CRC.

Evaluation of the Detrimental Impact of Low-Intensity Laser Radiation on the Characteristics of Sperm Movement, Motion, and DNA Damage

It is generally accepted that low-level laser therapy promotes cellular energy production and increases the synthesis of ATP by triggering the mitochondrial electron transport chain's photosensitive cytochrome c oxidase complex. Nevertheless, this investigation aimed to examine the possible adverse impact on sperm function and DNA integrity of low-intensity laser radiation with a wavelength of 980 nm. Following standard analysis, forty semen samples were collected and categorized as either asthenospermic, oligospermic, or normospermic. The remaining semen was subjected to standard semen analysis, and the aliquots were divided into treatment and control groups. A continuous-wave 980 nm laser with an output power of 100 mW and an energy density of 10 J/cm2 was applied to the treated samples for 30 s. Divided samples underwent incubation at 37oC, and semen analysis was used to assess aliquots at 30 s and 2 h intervals. Following the incubation time, each sample was frozen in 250 mL at -80oC until DNA fragmentation was examined using flow cytometry. Thirty minutes after treatment, there was a noticeable increase in sperm movement; the rise was largest in oligospermic and asthenospermic samples (88%) while samples of normal sperm displayed the least increment (7.5%). There was no discernible increase in DNA damage in the treatment samples compared to the control samples. The treatment samples had 21.8% and the control samples had 25.3% of the DNA fragmentation index. The dynamics of sperm motion were noticeably altered. Short-term exposure to low-level laser radiation appeared to improve the motion and movement of treated sperm, and after 2 h, there was no increase in DNA damage. Asthenospermia and mitochondrial dysfunction may be related in some circumstances. To fully understand the ramifications of these results for possible medicinal applications, more research is needed. Our research's conclusions suggest that low-level laser stimulation is a useful and safe technique for identifying viable immotile spermatozoa in a particular sample. It may also cause these spermatozoa to move about, which could have positive therapeutic effects.

Structural profiles of the full phagocyte NADPH oxidase unveiled by combining computational biology and experimental knowledge

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47phox, p40phox, p67phox, and Rac) with the membrane-bound flavocytochrome b558 (cytb558). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb558, whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47phox with cytb558, which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C-terminus of p47phox interacts with the cytb558. Combining the AF2 cytb558 -p47phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47phox, particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47phox, in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb558-Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multi-subunit assembly.

Analysis of the potential of the electroactive biofilm growth in a microbial fuel cell type H

Microbial fuel cells (MFC) constitute an attractive alternative as an environmental remediation technology since they can generate electrical current using organic waste as a substrate. Since the performance of MFCs depends on the characteristics of the biofilm on the anode surface, it is important to assess the genetic information of the microorganisms that grow on the electrode. For this purpose, a sewage sludge sample was obtained from a wastewater treatment plant and used to inoculate a type H MFC. Electrochemical characterization, on one hand, indicates that while the biofilm has a typical electrochemical performance reflected by the generated voltage (near 0.4 V) and by the electroactivity observed in cyclic voltammetry experiments, and on the other hand, the metagenomic analysis shows that the most abundant genera are Pseudomonacea, Nitrosomonas, Hyphomonas, and Opitutus. The study also indicates that the biofilm’s electroactive microorganisms can metabolize amino acids, lipids, and carbohydrates and possess genetic tools for ionic transport and energy production. Regarding the electron acceptor/donator capabilities, several oxidases, reductases, and complexes were identified, mainly terminal cytochrome C oxidase and respiratory complex I, which could be associated with the exoelectrogenic capacity of the microorganisms. Finally, the metagenomic information indicates that the biofilm can synthesize rhamnose, sialic acid, and alginate molecules, which could possibly be associated with the formation and consolidation of the microbial biofilm.

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana

ABSTRACT Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana’s pathogenic mechanisms.

Infected wound repair correlates with collagen I induction and NOX2 activation by cold atmospheric plasma

Cold atmospheric plasma (CAP) is a promising complement to tissue repair and regenerative medicine approaches. CAP has therapeutic potential in infected cutaneous wounds by mechanisms which remain enigmatic. Here, CAP is shown to activate phagocyte NADPH oxidase complex NOX2. CAP induced increased intracellular reactive oxygen species, alleviated by NOX2 inhibitors. Genetic and pharmacological inhibitions of NOX2 in macrophages and bioengineered skin infected with Staphylococcus aureus and treated with CAP reduced intracellular oxidants and increased bacterial survival. CAP triggered Rac activation and phosphorylation of p40phox and p47phox required for NOX2 assembly and activity. Furthermore, CAP induced collagen I expression by fibroblasts. Infection and healing kinetics showed that murine skin wounds infected with S. aureus and treated with CAP are characterized by decreased bacterial burden, increased length of neoepidermis and extracellular matrix formation. Collectively, our findings identify mechanisms triggered by CAP that subdue infection and result in enhanced repair following skin injury.

NoxO1 Determines the Level of ROS Formation by the Nox1-Centered NADPH Oxidase.

The Nox1-centered NADPH oxidase complex facilitates the transfer of electrons from intracellular NADPH across the cell membrane to extracellular molecular oxygen, resulting in the formation of superoxide. The complex is comprised of two membrane-bound subunits, namely Nox1 and p22phox, and the cytosolic subunits, namely NoxA1 and NoxO1. The presence of NoxO1 facilitates the proximity of all components, thereby enabling the complex to exhibit constitutive activity. Despite the theoretical sufficiency of all subunits in a 1:1 ratio, the precise composition of the Nox1-centered NADPH oxidase remains unknown. Analyses of mRNA expression in different cell lines revealed an unequal expression of the components, with an excess of NoxO1. Furthermore, plasmid-based overexpression of individual components of the Nox1-centered NADPH oxidase resulted in an excess of NoxO1 mRNA. The objective of this study was to analyze the ability of NoxO1 to control the level of ROS formation by the Nox1 complex. To this end, we generated Hek293 cells for constitutive expression of Nox1 and NoxA1, which were then transfected with increasing concentrations of NoxO1. The data presented herein suggests that ROS formation by the Nox1-centered NADPH oxidase is dependent on the concentration of NoxO1. A surplus of NoxO1 has been observed to exert control over the activity of the complex in accordance with a dose-dependent mechanism. We thus conclude that the ratio of Nox1, NoxA1, and NoxO1 complexes does not adhere to a 1:1 ratio. Conversely, the availability of NoxO1 serves to regulate the formation of ROS by the Nox1-centered NADPH oxidase.

UNVEILING THERAPEUTIC TARGETS THROUGH PATHWAY ANALYSIS AND IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN ULCERATIVE COLITIS

Objective: This study utilizes integrated bioinformatics to investigate Differentially Expressed Genes (DEGs) and pathways related to ulcerative colitis (UC). Material and Method: Differentially Expressed Genes were identified from UC patients' colonic mucosal samples and controls using GSE13367 and GSE134025 datasets. Differentially Expressed Genes selection utilized GEO2R and Venn diagrams, followed by functional annotation, pathway analysis, PPI determination via the STRING database, and GO/KEGG enrichment analysis using Metascape. Result and Discussion: Analysis unveiled 197 DEGs, with 76 up-regulated and 121 down-regulated genes. Up-regulated genes were enriched in humoral immune response, peptidoglycan binding, and NADPH oxidase complex, while down-regulated genes were linked to inorganic anion transport, transmitter-gated ion channel activity, and integral plasma membrane components. In the PPI network, up-regulated DEGs formed a dense network (75 nodes, 190 edges), indicating significant interactions, whereas down-regulated DEGs formed a less dense network (114 nodes, 63 edges). Five hub genes (CXCR4, CXCL13, CXCL1, MMP3) were identified among the 197 DEGs. These findings provide new insights into UC's causes and offer promise for more effective therapeutic approaches.

Abstract Tu128: p22 phox is a critical factor for the prevention of oxidation and stabilization of SERCA2a in the heart.

Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods/approach: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling. Results: The p22 phox -cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H 2 O 2 levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22 phox -cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22 phox -cKO mice. Furthermore, in the absence of p22 phox , SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22 phox knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22 phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Conclusion: The loss of p22 phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.

Chronic granulomatous disease: A single-center experience in Central Anatolia

BackgroundChronic granulomatous disease (CGD), one of the phagocytic cell defects, is the primary immunodeficiency caused by dysfunction of the NADPH oxidase complex in neutrophils. MethodsThe clinical, demographic and laboratory findings of 17 CGD patients who were followed-up between 2002 and 2021 were obtained retrospectively from the records of the patients. ResultsThe number of male and female patients was 10/7. The median age at diagnosis was 5.3 months (range 4–120) for 3 patients with X-CGD, and 42.4 months (range 8–350) for 14 patients with AR-CGD. We have investigated rare CYBA exon 3–6 deletion in 7 patients and hotspot mutation with delGT at the beginning of exon 2 of NCF1 in 5 patients. The most common clinical findings were pneumonia and lymphadenitis with recurrent fever, respectively (41.2%, 35.3%). A total of 154 microbial infections requiring hospital admission (27 in 3 XL and 127 in 14 AR patients) were detected in the follow-up of the patients and median infection number for a patient was 9 in both groups. Eight of 17 patients had stem cell transplantation and the survival rate was 87.5%. ConclusionsX-CGD patients are more rapidly recognized by family history and severe infections than those with AR-CGD and early prophylaxis may decrease infectious episodes. We have investigated the large deletion suggesting a possible founder effect for CYBA exon 3–6 deletion in Central Anatolia. Additionally, HSCT transplantation leads to a high survival rate for the patients with CGD.

A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

The level of blood neutrophils primary reactive oxygen species production in Graves’ disease patients undergoing conservative therapy with thiamazole.

Background. Conservative therapy with thiamazole in Graves’ disease helps to reduce the level of oxidative stress by restoring euthyroidism and the total reactive antioxidant potential of plasma. However, until now, in patients with Graves’ disease, the radical-producing ability of neutrophils remains unexplored at the initial stage of free radical conversion at different durations of drug-induced euthyroidism, which seems significant in revealing the mechanisms of disease recurrence. The aim is to study the level of production of primary reactive oxygen species in peripheral blood neutrophils depending on the duration of drug — induced euthyroidism in patients with Graves’ disease. Materials and methods. A single-center, prospective, controlled study was conducted with the participation of patients with laboratory-confirmed Graves’ disease. Subgroups of patients with Graves’ disease were formed depending on the duration of achieved drug-induced euthyroidism with continuous conservative therapy with thiamazole. The level of ROS production in peripheral blood neutrophils was determined by chemiluminescence. The assessment of spontaneous and zymosan-induced chemiluminescence was carried out for 90 minutes on a 36-channel chemiluminescence analyzer BLM-3607 (Medbiotech LLC, Krasnoyarsk). Results. The study included 102 Graves’ disease women with an average age of 47.54 ± 15.47 years. Of these, 75 (73.5 %) with relapse and 27 (26.47 %) with newly diagnosed disease. The total synthesis of (S) primary ROS increased statistically significantly relative to the control values in the examined patients with a duration of the euthyroid state of 5–8 and 9–12 months. It was found that in patients with Graves’ disease with stable drug-induced euthyroidism for 5–8 and 9–12 months, the S index of zymosan-induced lucigenin-dependent chemiluminescence of neutrophils increased statistically significantly, respectively, by 10.7 and 7.4 times relative to the control values. Conclusion. The restoration and maintenance of drug-induced euthyroidism for more than 12 months against the background of conservative thiamazole therapy in patients with Graves’ disease reduces the load of systemic oxidative stress on the immune system. However, the high total production of primary ROS in neutrophils of patients with Graves’ disease in a state of drug-induced euthyroidism with conservative thiamazole therapy may play a significant role in disconnecting the functioning of the NADPH oxidase complex and determine the functional capabilities of neutrophils in case of disease recurrence.

Влияние ингибиторов фенолоксидаз на эффективность деколоризации малахитового зеленого бактериями рода Azo spirillum

Synthetic dyes are widely used in various branches of light industry. Due to the insufficient efficiency of industrial painting processes, a large percentage of dyes end up in the wastewater of enterprises in an unmodified form, which creates a huge risk of environmental pollution with these compounds. Triphenylmethane dyes, in particular malachite green, are toxic, allergenic and carcinogenic compounds. To date, biodegradability of triphenylmethane dyes has been shown for some bacteria and fungi producing phenol oxidase complex enzymes, including soil associative bacteria of the genus Azospirillum. Many factors are capable of inducing and inhibiting the biodegradation efficiency, in particular the enzymatic systems that are involved in bleaching processes. In the present work we studied the effects of typical deactivating agents of phenol oxidases, such as H2O2, EDTA, SDS-Na, β-mercaptoethanol, dithiothreitol, Tween, and sodium azide, on the azospirilla’s phenol oxidases activity and the ability to decolorize malachite green. It was found that Tween and sodium azide do not have an inhibitory effect on azospirillum enzymes and exhibit a total stabilizing effect on the entire complex. An inhibitory effect from 60 to 100% was noted for laccase and Mn-peroxidase activity under the action of β-mercaptoethanol, dithiothreitol and EDTA, which is directly proportional to the decolorization rate of malachite green. Compared with the latest issues on classical phenol-oxidizing enzymes, our data revealed non-typical properties of the phenol oxidase complex enzymes of azospirillum.

Sls1 and Mtf2 mediate the assembly of the Mrh5C complex required for activation of cox1 mRNA translation

Mitochondrial translation depends on mRNA-specific activators. In Schizosaccharomyces pombe, DEAD-box protein Mrh5, pentatricopeptide repeat (PPR) protein Ppr4, Mtf2, and Sls1 form a stable complex (designated Mrh5C) required for translation of mitochondrial DNA (mtDNA)‐encoded cox1 mRNA, the largest subunit of the cytochrome c oxidase complex. However, how Mrh5C is formed and what role Mrh5C plays in cox1 mRNA translation have not been reported. To address these questions, we investigated the role of individual Mrh5C subunits in the assembly and function of Mrh5C. Our results revealed that Mtf2 and Sls1 form a subcomplex that serves as a scaffold to bring Mrh5 and Ppr4 together. Mrh5C binds to the small subunit of the mitoribosome (mtSSU), but each subunit could not bind to the mtSSU independently. Importantly, Mrh5C is required for the association of cox1 mRNA with the mtSSU. Finally, we investigated the importance of the signature DEAD-box in Mrh5. We found that the DEAD-box of Mrh5 is required for the association of Mrh5C and cox1 mRNA with the mtSSU. Unexpectedly, this motif is also required for the interaction of Mrh5 with other Mrh5C subunits. Altogether, our results suggest that Mrh5 and Ppr4 cooperate in activating the translation of cox1 mRNA. Our results also suggest that Mrh5C activates the translation of cox1 mRNA by promoting the recruitment of cox1 mRNA to the mtSSU.

Functional Conservation of the Small GTPase Rho5/Rac1-A Tale of Yeast and Men.

Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.

ROS signaling in innate immunity via oxidative protein modifications.

The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O2 •-) that functions as a precursor for antimicrobial hydrogen peroxide (H2O2) production, and H2O2 is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. H2O2 modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.