Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor.

BACKGROUND: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor. RESULTS: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869. CONCLUSION: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

RESISTANCE TO PHYTOPHTHORA1 promotes cytochrome b559 formation during early photosystem II biogenesis in Arabidopsis.

As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in the protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of reactive oxygen species under photoinhibitory-light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.

The role of Listeria monocytogenes PstA in ß-lactam resistance requires the cytochrome bd oxidase activity.

c-di-AMP is an essential second messenger that binds and regulates several proteins of different functions within bacterial cells. Among those, PstA is a structurally conserved c-di-AMP-binding protein, but its function is largely unknown. PstA is structurally similar to PII signal transduction proteins, although it specifically binds c-di-AMP rather than other PII ligands such as ATP and α-ketoglutarate. In Listeria monocytogenes, we found that PstA increases ß-lactam susceptibility at normal and low c-di-AMP levels, but increases ß-lactam resistance upon c-di-AMP accumulation. Examining a PstA mutant defective for c-di-AMP binding, we found the apo form of PstA to be toxic for ß-lactam resistance, and the c-di-AMP-bound form to be beneficial. Intriguingly, a role for PstA in ß-lactam resistance is only prominent in aerobic cultures, and largely diminished under hypoxic conditions, suggesting that PstA function is linked to aerobic metabolism. However, PstA does not control aerobic growth rate, and has a modest influence on the tricarboxylic acid cycle and membrane potential-an indicator of cellular respiration. The regulatory role of PstA in ß-lactam resistance is unrelated to reactive oxygen species or oxidative stress. Interestingly, during aerobic growth, PstA function requires the cytochrome bd oxidase (CydAB), a component of the respiratory electron transport chain. The requirement for CydAB might be related to its function in maintaining a membrane potential, or redox stress response activities. Altogether, we propose a model in which apo-PstA diminishes ß-lactam resistance by interacting with an effector protein, and this activity can be countered by c-di-AMP binding or a by-product of redox stress. IMPORTANCE: PstA is a structurally conserved c-di-AMP-binding protein that is broadly present among Firmicutes bacteria. Furthermore, PstA binds c-di-AMP at high affinity and specificity, indicating an important role in the c-di-AMP signaling network. However, the molecular function of PstA remains elusive. Our findings reveal contrasting roles of PstA in ß-lactam resistance depending on c-di-AMP-binding status. We also define physiological conditions for PstA function during aerobic growth. Future efforts can exploit these conditions to identify PstA interaction partners under ß-lactam stress.

Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB.

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s-1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

Structural basis for an early stage of the photosystem II repair cycle in Chlamydomonas reinhardtii.

Photosystem II (PSII) catalyzes water oxidation and plastoquinone reduction by utilizing light energy. It is highly susceptible to photodamage under high-light conditions and the damaged PSII needs to be restored through a process known as the PSII repair cycle. The detailed molecular mechanism underlying the PSII repair process remains mostly elusive. Here, we report biochemical and structural features of a PSII-repair intermediate complex, likely arrested at an early stage of the PSII repair process in the green alga Chlamydomonas reinhardtii. The complex contains three protein factors associated with a damaged PSII core, namely Thylakoid Enriched Factor 14 (TEF14), Photosystem II Repair Factor 1 (PRF1), and Photosystem II Repair Factor 2 (PRF2). TEF14, PRF1 and PRF2 may facilitate the release of the manganese-stabilizing protein PsbO, disassembly of peripheral light-harvesting complexes from PSII and blockage of the QB site, respectively. Moreover, an α-tocopherol quinone molecule is located adjacent to the heme group of cytochrome b559, potentially fulfilling a photoprotective role by preventing the generation of reactive oxygen species.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects.

Cytochrome b561 (cytb561) proteins comprise a family of transmembrane oxidoreductases that transfer single electrons across a membrane. Most eukaryotic species, including insects, possess multiple cytb561 homologs. To learn more about this protein family in insects, we carried out a bioinformatics-based investigation of cytb561 family members from nine species representing eight insect orders. We performed a phylogenetic analysis to classify insect cytb561 ortholog groups. We then conducted sequence analyses and analyzed protein models to predict structural elements that may impact the biological functions and localization of these proteins, with a focus on possible ferric reductase activity. Our study revealed three ortholog groups, designated CG1275, Nemy, and CG8399, and a fourth group of less-conserved genes. We found that CG1275 and Nemy proteins are similar to a human ferric reductase, duodenal cytochrome b561 (Dcytb), and have many conserved amino acid residues that function in substrate binding in Dcytb. Notably, CG1275 and Nemy proteins contain a conserved histidine and other residues that play a role in ferric ion reduction by Dcytb. Nemy proteins were distinguished by a novel cysteine-rich cytoplasmic loop sequence. CG8399 orthologs are similar to a putative ferric reductase in humans, stromal cell-derived receptor 2. Like other members of the CYBDOM class of cytb561 proteins, these proteins contain reeler, DOMON, and cytb561 domains. Drosophila melanogaster CG8399 is the only insect cytb561 with known ferric reductase activity. Our investigation of the DOMON domain in CG8399 proteins revealed a probable heme-binding site and a possible site for ferric reduction. The fourth group includes a subgroup of proteins with a conserved "KXXXXKXH" non-cytoplasmic loop motif that may be a substrate binding site and is present in a potential ferric reductase, human tumor suppressor cytochrome b561. This study provides a foundation for future investigations of the biological functions of cytb561 genes in insects.

Rubredoxin 1 promotes the proper folding of D1 and is not required for heme b559 assembly in Chlamydomonas photosystem II.

Photosystem II (PSII), the water:plastoquinone oxidoreductase of oxygenic photosynthesis, contains a heme b559 iron whose axial ligands are provided by histidine residues from the α (PsbE) and ß (PsbF) subunits. PSII assembly depends on accessory proteins that facilitate the step-wise association of its protein and pigment components into a functional complex, a process that is challenging to study due to the low accumulation of assembly intermediates. Here, we examined the putative role of the iron[1Fe-0S]-containing protein rubredoxin 1 (RBD1) as an assembly factor for cytochrome b559, using the RBD1-lacking 2pac mutant from Chlamydomonas reinhardtii, in which the accumulation of PSII was rescued by the inactivation of the thylakoid membrane FtsH protease. To this end, we constructed the double mutant 2pac ftsh1-1, which harbored PSII dimers that sustained its photoautotrophic growth. We purified PSII from the 2pac ftsh1-1 background and found that α and ß cytochrome b559 subunits are still present and coordinate heme b559 as in the WT. Interestingly, immunoblot analysis of dark- and low light-grown 2pac ftsh1-1 showed the accumulation of a 23-kDa fragment of the D1 protein, a marker typically associated with structural changes resulting from photodamage of PSII. Its cleavage occurs in the vicinity of a nonheme iron which binds to PSII on its electron acceptor side. Altogether, our findings demonstrate that RBD1 is not required for heme b559 assembly and point to a role for RBD1 in promoting the proper folding of D1, possibly via delivery or reduction of the nonheme iron during PSII assembly.

DDX17 induces epithelial-mesenchymal transition and metastasis through the miR-149-3p/CYBRD1 pathway in colorectal cancer.

DEAD box helicase 17 (DDX17) has been reported to be involved in the initiation and development of several cancers. However, the functional role and mechanisms of DDX17 in colorectal cancer (CRC) malignant progression and metastasis remain unclear. Here, we reported that DDX17 expression was increased in CRC tissues compared with noncancerous mucosa tissues and further upregulated in CRC liver metastasis compared with patient-paired primary tumors. High levels of DDX17 were significantly correlated with aggressive phenotypes and worse clinical outcomes in CRC patients. Ectopic expression of DDX17 promoted cell migration and invasion in vitro and in vivo, while the opposite results were obtained in DDX17-deficient CRC cells. We identified miR-149-3p as a potential downstream miRNA of DDX17 through RNA sequencing analysis, and miR-149-3p displayed a suppressive effect on the metastatic potential of CRC cells. We demonstrated that CYBRD1 (a ferric reductase that contributes to dietary iron absorption) was a direct target of miR-149-3p and that miR-149-3p was required for DDX17-mediated regulation of CYBRD1 expression. Moreover, DDX17 contributed to the metastasis and epithelial to mesenchymal transition (EMT) of CRC cells via downregulation of miR-149-3p, which resulted in increased CYBRD1 expression. In conclusion, our findings not only highlight the significance of DDX17 in the aggressive development and prognosis of CRC patients, but also reveal a novel mechanism underlying DDX17-mediated CRC cell metastasis and EMT progression through manipulation of the miR-149-3p/CYBRD1 pathway.

pH-dependent kinetics of NO release from E. coli bd-I and bd-II oxidase reveals involvement of Asp/Glu58B.

Escherichia coli contains two cytochrome bd oxidases, bd-I and bd-II. The structure of both enzymes is highly similar, but they exhibit subtle differences such as the accessibility of the active site through a putative proton channel. Here, we demonstrate that the duroquinol:dioxygen oxidoreductase activity of bd-I increased with alkaline pH, whereas bd-II showed a broad activity maximum around pH 7. Likewise, the pH dependence of NO release from the reduced active site, an essential property of bd oxidases, differed between the two oxidases as detected by UV/vis spectroscopy. Both findings may be attributed to differences in the proton channel leading to the active site heme d. The channel comprises a titratable residue (Asp58B in bd-I and Glu58B in bd-II). Conservative mutations at this position drastically altered NO release demonstrating its contribution to the process.

Cytochrome bd-I from Escherichia coli is catalytically active in the absence of the CydH subunit.

Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.

E. coli cytochrome bd-I requires Asp58 in the CydB subunit for catalytic activity.

The reduction of oxygen to water is crucial to life and a central metabolic process. To fulfil this task, prokaryotes use among other enzymes cytochrome bd oxidases (Cyt bds) that also play an important role in bacterial virulence and antibiotic resistance. To fight microbial infections by pathogens, an in-depth understanding of the enzyme mechanism is required. Here, we combine bioinformatics, mutagenesis, enzyme kinetics and FTIR spectroscopy to demonstrate that proton delivery to the active site contributes to the rate limiting steps in Cyt bd-I and involves Asp58 of subunit CydB. Our findings reveal a previously unknown catalytic function of subunit CydB in the reaction of Cyt bd-I.

Heme A Synthase Deficiency Affects the Ability of Bacillus cereus to Adapt to a Nutrient-Limited Environment.

The branched aerobic respiratory chain in Bacillus cereus comprises three terminal oxidases: cytochromes aa3, caa3, and bd. Cytochrome caa3 requires heme A for activity, which is produced from heme O by heme A synthase (CtaA). In this study, we deleted the ctaA gene in B. cereus AH187 strain, this deletion resulted in loss of cytochrome caa3 activity. Proteomics data indicated that B. cereus grown in glucose-containing medium compensates for the loss of cytochrome caa3 activity by remodeling its respiratory metabolism. This remodeling involves up-regulation of cytochrome aa3 and several proteins involved in redox stress response-to circumvent sub-optimal respiratory metabolism. CtaA deletion changed the surface-composition of B. cereus, affecting its motility, autoaggregation phenotype, and the kinetics of biofilm formation. Strikingly, proteome remodeling made the ctaA mutant more resistant to cold and exogenous oxidative stresses compared to its parent strain. Consequently, we hypothesized that ctaA inactivation could improve B. cereus fitness in a nutrient-limited environment.

Tandem gene amplification restores photosystem II accumulation in cytochrome b559 mutants of cyanobacteria.

Cytochrome (Cyt) b559 is a key component of the photosystem II complex (PSII) that is essential for its proper functioning and assembly. Site-directed mutants of the model cyanobacterium Synechocystis sp. PCC6803 with mutated heme axial ligands of Cyt b559 have little PSII and are therefore unable to grow photoautotrophically. Here we describe two types of Synechocystis autotrophic transformants that retained the same mutations in Cyt b559 but are able to accumulate PSII and grow photoautotrophically. Whole-genome sequencing revealed that all of these autotrophic transformants carried a variable number of tandem repeats (from 5 to 15) of chromosomal segments containing the psbEFLJ operon. RNA-seq analysis showed greatly increased transcript levels of the psbEFLJ operon in these autotrophic transformants. Multiple copies of the psbEFLJ operon in these transformants were only maintained during autotrophic growth, while its copy numbers gradually decreased under photoheterotrophic conditions. Two-dimensional PAGE analysis of membrane proteins revealed a strong deficiency in PSII complexes in the Cyt b559 mutants that was reversed in the autotrophic transformants. These results illustrate how tandem gene amplification restores PSII accumulation and photoautotrophic growth in Cyt b559 mutants of cyanobacteria, and may serve as an important adaptive mechanism for cyanobacterial survival.

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

miR-423-3p activates FAK signaling pathway to drive EMT process and tumor growth in lung adenocarcinoma through targeting CYBRD1.

BACKGROUND: Lung adenocarcinoma (LUAD) is a malignant tumor with a high fatality rate and poor overall survival, while molecular targets diagnosing and alleviating lung cancer remain inadequate. METHODS: In this article, we highlighted the upregulation of microRNA-423-3p (miR-423-3p) in LUAD, especially in smokers aged over 40, and revealed that the high expression of miR-423-3p was significantly associated with smoker, age, and pathologic stage of LUAD patients. RESULTS: Moreover, overexpressing miR-423-3p could facilitate LUAD cell proliferation, invasion, adhesion, and epithelial-mesenchymal transition (EMT) process, while depleted miR-423-3p caused repressive influence upon it. Mechanically, we identified that miR-423-3p could activate FAK signaling pathway through binding to the 3'-UTR of cytochrome B reductase 1 (CYBRD1). Furthermore, we demonstrated that CYBRD1 was lowly expressed in LUAD, and miR-423-3p overexpression could rescue the impairment of LUAD cell proliferation, invasion, adhesion, and EMT caused by CYBRD1 depletion. Noticeably, miR-423-3p depletion efficiently hindered LUAD tumor growth in vivo. CONCLUSION: Collectively, our findings demonstrated that miR-423-3p/CYBRD1 axis could be regarded as a promising biomarker to alleviate the poor LUAD prognosis.

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state.

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. RESULTS: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. CONCLUSION: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Granuloma Formation in a Cyba-Deficient Model of Chronic Granulomatous Disease Is Associated with Myeloid Hyperplasia and the Exhaustion of B-Cell Lineage.

Haematopoiesis is a paradigm of cell differentiation because of the wide variety and overwhelming number of mature blood cells produced daily. Under stress conditions, the organism must adapt to a boosted demand for blood cells. Chronic granulomatous disease (CGD) is a genetic disease caused by inactivating mutations that affect the phagocyte oxidase. Besides a defective innate immune system, CGD patients suffer from recurrent hyper-inflammation episodes, circumstances upon which they must face emergency haematopoiesis. The targeting of Cybb and Ncf1 genes have produced CGD animal models that are a useful surrogate when studying the pathophysiology and treatment of this disease. Here, we show that Cyba-/- mice spontaneously develop granuloma and, therefore, constitute a CGD animal model to complement the existing Cybb-/- and Ncf1-/- models. More importantly, we have analysed haematopoiesis in granuloma-bearing Cyba-/- mice. These animals showed a significant loss of weight, developed remarkable splenomegaly, bone marrow myeloid hyperplasia, and signs of anaemia. Haematological analyses showed a sharped decrease of B-cells and a striking development of myeloid cells in all compartments. Collectively, our results show that granuloma inflammatory lesions dramatically change haematopoiesis homeostasis. Consequently, we suggest that besides their defective innate immunity, the alteration of haematopoiesis homeostasis upon granuloma may contribute to the dismal outcome of CGD.

Carnosic Acid Attenuates an Early Increase in ROS Levels during Adipocyte Differentiation by Suppressing Translation of Nox4 and Inducing Translation of Antioxidant Enzymes.

The objective of this study was to investigate molecular mechanisms underlying the ability of carnosic acid to attenuate an early increase in reactive oxygen species (ROS) levels during MDI-induced adipocyte differentiation. The levels of superoxide anion and ROS were determined using dihydroethidium (DHE) and 2'-7'-dichlorofluorescin diacetate (DCFH-DA), respectively. Both superoxide anion and ROS levels peaked on the second day of differentiation. They were suppressed by carnosic acid. Carnosic acid attenuates the translation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4), p47phox, and p22phox, and the phosphorylation of nuclear factor-kappa B (NF-κB) and NF-κB inhibitor (IkBa). The translocation of NF-κB into the nucleus was also decreased by carnosic acid. In addition, carnosic acid increased the translation of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCSc), and glutathione S-transferase (GST) and both the translation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, these results indicate that carnosic acid could down-regulate ROS level in an early stage of MPI-induced adipocyte differentiation by attenuating ROS generation through suppression of NF-κB-mediated translation of Nox4 enzyme and increasing ROS neutralization through induction of Nrf2-mediated translation of phase II antioxidant enzymes such as HO-1, γ-GCS, and GST, leading to its anti-adipogenetic effect.