Cytochrome B Research Articles

Combined effect of microplastics and tire particles on Daphnia magna: Insights from physiological and transcriptomic responses.

Abstract Background and Aims The kidney and the brain share similarities in terms of structure and function, and are coordinated by common pathogenic mechanisms, such as inflammation, endothelial dysfunction, oxidative stress, and mitochondrial dysfunction. The aim of our study was to evaluate the interrelation of these pathogenic pathways with proximal tubule (PT) dysfunction, podocyte damage, and cerebral vessels remodeling in normoalbuminuric DKD in type 2 diabetes mellitus (DM) patients. Method A cohort of 223 patients (64 patients with normoalbuminuria, 59 patients with microalbuminuria, and 61 patients with macroalbuminuria) and 39 age- and gender-matched healthy controls were enrolled in this case series study. All patients were assessed concerning urinary albumin/creatinine ratio (UACR), biomarkers of podocyte damage (synaptopodin, podocalyxin) and of proximal tubule dysfunction (kidney injury molecule-1-KIM-1, N-acetyl-β-(D)-glucosaminidase-NAG), serum and urinary stromal cell-derived factor-1 (SDF-1), P-selectin, advanced oxidation protein products (AOPPs). MtDNA-CN and nuclear DNA (nDNA) were quantified in peripheral blood and urine by qRT-PCR (ABI 7900-HT–Applied BioSystem). TaqMan assays were utilized for the assessment of cytochrome b (CYTB) gene, subunit 2 of NADH dehydrogenase (ND2), and of beta 2 microglobulin nuclear gene (B2M). mtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies, through analysis of the CYTB/B2M and ND2/B2M ratio. The cerebrovascular hemodynamic indices were evaluated by an ultrasound equipment with high resolution (MYLAB 8-ESAOTE Italy) equipped with a colour ultrasound system. Subclinical atherosclerosis and arteriosclerosis were assessed by intima-media thickness (IMT) in the common carotid arteries (CCAs), the pulsatility index (PI) and the resistivity index (RI) in the internal carotid arteries (ICAs) and middle cerebral arteries (MCAs), bilaterally. Cerebrovascular reactivity (CVR), which represents the vasodilatory reactivity of cerebral vessels in response to a vasodilatory stimulus, was assessed by the breath-holding test and the breath-holding index (BHI). Results In multivariable regression analysis, serum mtDNA correlated directly with eGFR and negatively with serum P-selectin and serum SDF-1 (R2 = 0.6564; P < 0.0001). Urinary mtDNA correlated directly with podocalyxin, urinary P-selectin, and urinary SDF-1 (R2 = 0.6280; P < 0.0001). IMT-CCAs correlated negatively with eGFR and directly with UACR, synaptopodin, serum P-selectin, and serum AOPPs (P < 0.0001; R2 = 0.7020). PI-ICAs correlated indirectly with eGFR and directly with serum P-selectin and serum SDF-1 (P < 0.0001; R2 = 0.5113). PI-MCAs had a negative correlation with eGFR and direct correlations serum P-selectin and serum SDF-1(P < 0.0001; R2 = 0.5886). RI-ICAs was included in a complex model which displayed an indirect correlation with eGFR and direct correlations with NAG, serum P-selectin, serum SDF-1, and UACR (P < 0.0001; R2 = 0.7629). RI-MCAs were included in a model which showed a negative correlation with eGFR and direct correlations with NAG, KIM-1, synaptopodin, podocalyxin, serum P-selectin and serum SDF-1 (P < 0.0001; R2 = 0.8482). The CVR the BHI correlated negatively with eGFR and directly with podocalyxin, serum P-selectin, and serum SDF-1 (P < 0.0001; R2 = 0.7897). Conclusion The study demonstrates a concurrent association of SDF-1, P-selectin, AOPPs, and mitochondrial dysfunction with early DKD and significant cerebrovascular modifications in neurologically asymptomatic patients with type 2 DM and normoalbuminuric DKD.

The cytochrome-b5 reductases (CYB5Rs) regulate cellular redox balance and contribute to the pathogenesis of inflammatory diseases. However, the roles of CYB5R5 in macrophages remain poorly understood and require further elucidation. In this study, we revealed that CYB5R5 orchestrates macrophage inflammation by inhibiting interleukin (IL)-1β production from M1 macrophages. Mechanistically, CYB5R5 enhances flavin adenine dinucleotide (FAD)-lysine demethylase1 (LSD1) signaling to regulate the histone demethylation of complement component 1, q subcomponent (C1q)-coding genes, thereby lowering NLRP3 inflammasome assembly. We also found that myeloid depletion of Cyb5r5 in mice exacerbates inflammatory responses in LPS-induced sepsis. This study reveals that CYB5R5 attenuates M1 macrophage polarization via metabolic and epigenetic reprogramming mechanism, thus providing potential therapeutic targets for macrophage-mediated inflammatory disorders.

Background/Objectives: Cytochrome 1B1 (CYP1B1) is overexpressed in several cancers, contributing to carcinogenesis, cancer progression, and chemoresistance. Despite its known oncogenic role, its expression in bone sarcomas remains unknown. Methods: This study assessed CYP1B1 expression in osteosarcoma and chondrosarcoma using immunohistochemistry on tissue microarrays and analyzed corresponding transcriptomic profiles from public RNA-seq datasets. Associations with clinicopathological features, survival, drug sensitivity, and protein–protein interaction networks were also investigated. Results: CYP1B1 was overexpressed in 72.3% of bone sarcomas (78% of osteosarcomas and 82.1% of chondrosarcomas) and was significantly underexpressed in normal bone (12.5%, p < 0.001). Importantly, high CYP1B1 expression was found in younger patients (≤34 years; p = 0.013), but no other associations with tumor grade, size, or metastasis were observed. The mean survival rate of CYP1B1-positive patients was insignificantly shorter than that of negative patients (58.8 vs. 62.8 months; p = 0.170). Although not confirmed in the multivariate analysis, CYP1B1-positive patients had poorer survival in the univariate analysis, which may reflect tumor aggressiveness rather than prognostic value. Transcriptomic data showed significantly lower CYP1B1 mRNA in osteosarcoma versus normal bone, suggesting post-transcriptional or translational regulation. Drug sensitivity analysis revealed both positive and negative correlations between CYP1B1 expression and response to various compounds in the GDSC dataset, highlighting potential therapeutic implications. Conclusions: Despite low mRNA levels, CYP1B1 protein is consistently and selectively overexpressed in bone sarcomas, particularly in younger patients. While not prognostic, its expression profile warrants further investigation and evaluation as a therapeutic target or diagnostic biomarker, especially in refractory or advanced cases.

Haplotype-based association of CYB5A gene polymorphisms (rs1790834 and rs1790858) with polycystic ovary syndrome in a south Indian cohort.

Fenofibrate Ameliorates Ocular Surface Inflammation in Diabetic Keratopathy.

One of the cytochrome b561 family members in C. elegans, named Cecytb-2, was investigated. Purified recombinant Cecytb-2 showed typical visible absorption spectra, EPR signals, and redox midpoint potentials, very similar to those of human Dcytb, which is responsible for intestinal iron acquisition by its ferric reductase activity. Fast kinetic experiments using pulse radiolysis and stopped-flow techniques showed that Cecytb-2 donates electrons to monodehydroascorbate radicals with a much lower reactivity than other cytochrome b561 members, but it can accept electrons from ascorbate (AsA) as rapidly as other members. DEPC treatment of Cecytb-2 caused significant inhibition of electron acceptance from AsA and lowered the midpoint potential of heme bL. MS/MS MASCOT analyses verified that N-carbethoxylations of conserved Lys98 and heme bL axial His101 residues on the cytosolic side were major causes of the inhibition. Reconstituted Cecytb-2 in sealed vesicle membranes, in which AsA was entrapped, showed significant transmembrane ferric reductase activity. In situ hybridization analysis revealed that Cecytb-2 mRNA was distributed in intestinal cells. Immunohistochemical analysis indicated that Cecytb-2 resided in intestinal lumens. Knockdown of the Cecytb-2 gene expression in N2 worms indicated a significant suppression of growth under ferrous ion-deficient conditions. Thus, the ferric reductase activity conferred by Cecytb-2 seems to participate in iron acquisition and is very important for normal growth in low-ferrous conditions, confirming that Cecytb-2 is a genuine Dcytb homolog in C. elegans.

ABSTRACT Streptococcus pneumoniae is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo. Using a transgenic macroautophagy/autophagy reporter line, we found the autophagy marker Map1lc3/Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy gene atg5 led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae-infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that the pneumococcal pore-forming toxin pneumolysin induces ROS-independent CASM pathways, one of which is abolished by knockdown of tecpr1a indicating the involvement of sphingomyelin-Tecpr1-induced LC3 lipidation (STIL). Collectively, our observations shed new light on the host immune response against S. pneumoniae, demonstrating that several distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections. Abbreviations: ATG: autophagy related; BMDM: bone marrow-derived macrophage; CASM: conjugation of ATG8 to single membranes; CFU: colony-forming units; Cyba: cytochrome b-245, alpha polypeptide; DPI: diphenyleneiodonium, GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LAP: LC3-associated phagocytosis; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NADPH: nicotinamide adenine dinucleotide phosphate; Optn: optineurin; PINCA: pore-forming toxin-induced non-canonical autophagy; Ply: pneumolysin; ROS: reactive oxygen species; SLR: sequestosome-like receptors; Sqstm1: sequestosome 1; STIL: sphingomyelin-TECPR1-induced LC3 lipidation; Tecpr1: tectonin beta-propeller repeat containing 1.

Cytochrome P450 comprises a group of monooxygenases that hydroxylate xenobiotics and natural compounds with diverse electron transfer systems. Here we identify a natural fusion protein of cytochrome (Cyt) b5 and Cyt b5 reductase (CBBR) that transfers electrons from NADH to the cytochrome P450 CYP540A2. This cytochrome P450 system hydroxylates medium-chain fatty acids (MCFAs) to generate (R)-β-hydroxy-MCFAs with 7-12 carbon atoms. Kinetic studies of CYP540A2 mutants indicate that side chains of Ser431 and Gln542 residues bind the carboxyl moiety of MCFAs for hydroxylation at their β-carbons. Pre-steady state kinetics also indicate that a predicted linker region between the FAD- and Cyt b5-domains of CBBR modulates electron transfer from NADH to CYP540A2. The present study also identifies a dehydrogenase that oxidizes (R)-β-hydroxy-MCFAs to β-oxo-fatty acids that are substrates in the general β-oxidation mechanism of fatty acid degradation. The genes encoding CBBR, CYP540A2, and (R)-β-hydroxy-MCFA dehydrogenase are clustered in the genome of the fungus Aspergillus nidulans and related fungi. The A. nidulans genes are induced by MCFAs, and disrupting CBBR and CYP540A2 genes accumulated more intracellular decanoic acid. Our findings reveal an adaptive monooxygenase-dependent β-oxidation mechanism that alternates with conventional β-oxidation, thus allowing fungi to metabolize MCFAs.

Genome-wide identification of cytochrome b5 gene family reveals their potential roles in nitrate response in Xanthium strumarium.

Background. Oxidative stress is crucial in the pathogenesis of endogenous intoxication syndrome in acute surgical abdominal pathology. The enzyme systems of oxidative phosphorylation and cytochrome reductase are vital for hepatocyte energy potential. This study aimed to investigate the effects of a p38 MAPK inhibitor on cytochrome b5 reductase 3 (CYB5R3); cytochrome c oxidase; and microsomal oxidation in experimental peritonitis.Methods. A model of peritonitis was used with two groups: a control group without treatment (n = 15) and a main group treated with adezmapimod conjugate on endof-surgery (n = 15). Immunomorphological staining was employed to evaluate CYB5R3 and cytochrome oxidase expression on days 3; 7; and 14. The intensity of staining was scored from 0 to 4; and differences were assessed using multiple comparison tests and the Mann-Whitney criterion.Results. In the control group; CYB5R3 intensity decreased from 2.5 [2.0;3.0] on day 3 to 1.0 [1.0;1.0] on day 14. Conversely; in the main group; the inhibitor maintained high enzyme expression throughout the study. On day 3; cytochrome oxidase levels differed significantly between groups: 1.0 [1.0;2.0] in the control and 4.0 [3.0;4.0] in the main group (p &lt; 0.05).Conclusion. The findings suggest that adezmapimod conjugate positively affects the integrity of enzyme systems; maintaining redox balance and oxidative phosphorylation in hepatocytes during peritonitis.

Cytoglobin and Neuroglobin are heme-containing proteins expressed in most vertebrates, including mammals, with still not completely defined physiological roles. Most of the putative functions of cytoglobin/neuroglobin, such as oxygen binding or nitric oxide dioxygenation, rely on the heme iron being in the ferrous (Fe2+) oxidation state. Therefore, it is very possible that reducing systems are active in the cell to maintain both proteins in the ferrous state. We have previously shown that the cytochrome b5 reductase isoform 3/ cytochrome b5 system, the canonical reductase of hemoglobin and myoglobin, can reduce cytoglobin at very fast rates, consistent with a possible physiological role. However this reducing system is unable to reduce neuroglobin, which to date lacks a validated, physiologically feasible reducing system.Here we have studied the interaction of cytochrome b5 reductase isoform 4 with cytoglobin and neuroglobin and found that cytochrome b5 reductase 4 can reduce cytoglobin at rates comparable to those observed with cytochrome b5 reductase 3/ cytochrome b5. Remarkably, it can also reduce neuroglobin efficiently. Studying different surface mutations of cytoglobin and neuroglobin we note that some cytoglobin mutations, in particular R84E and K116E decrease reduction rates by more than 10-fold, whereas surface mutations in neuroglobin that were shown to impair the interaction of neuroglobin with cytochrome c (E60K/D73K/E87K) show little effect on the reduction rates. We conclude that cytochrome b5 reductase 4 can supplement cytochrome b5 reductase 3/ cytochrome b5 roles for cytoglobin reduction in vivo and is a strong candidate for a physiological role as neuroglobin reductase.

Reusable and modular combinatorial libraries for iterative metabolic engineering of Saccharomyces cerevisiae.

Sterols are essential isoprenoid derivatives that contribute to membrane structure and function. In plants, they also serve as precursors to phytohormones and specialized metabolites important for development, defense, and health. Although the sterol biosynthetic pathway is considered well-characterized, we report the discovery of a plant-specific cytochrome b5-like protein, CB5LP, as a critical component of phytosterol biosynthesis. Loss of CB5LP in Arabidopsis causes embryonic defects, seedling lethality, and accumulation of 14α-methyl-sterols, with reduced levels of sitosterol and stigmasterol-indicating a defect in sterol 14α-demethylation. TurboID-based proximity labeling and in vitro assays show that CB5LP physically and functionally interacts with CYP51, a cytochrome P450 enzyme catalyzing this demethylation step. Unlike canonical cytochrome b5 proteins, CB5LP has a reversed topology and is exclusive to plants, acting as an evolutionarily distinct electron donor. This discovery reveals an uncharacterized redox partnership essential for sterol biosynthesis and highlights a promising target for the development of selective herbicide.

Association analysis reveals SNP markers associated with growth traits in swimming crabs (Portunus trituberculatus).

Genome-wide identification and functional analysis of SmCB5 genes in eggplant highlights SmCB5-3 as a major player in anthocyanin production.

Polyphagous fruit fly (Diptera: Tephritidae) pests from the Pacific Islands pose a biosecurity risk to New Zealand, a country free from pest fruit flies. Among them, Bactrocera facialis, B. passiflorae, B. kirki, and B. distincta are sympatric species commonly intercepted at immature stages at the border. However, current mitochondrial cytochrome oxidase I (COI) barcode data lack sufficient variation for a confident identification of the above four species. To address this, we generated COI barcode data for 403 fruit fly individuals including these four species and an additional 17 related fruit fly species. A phylogenetic analysis of the COI sequences of B. facialis revealed two genetically distinct populations, one closely related to B. passiflorae. Complete mitochondrial genomes were explored, identifying minor fixed differences in Cytochrome b (CYTB), NADH dehydrogenase 2 (ND2), and ATP synthase membrane subunit 6 (ATP6) genes. Based on sequence data for COI, ND2, and ATP6 genes, a multiplex real-time PCR assay has been developed and validated for the four target species. Each assay demonstrated high specificity, with no cross-reactions, and sensitivity as low as 10 copies/μL of the target DNA. This study shows that the developed assays enable the rapid and reliable identification of the target fruit fly species, supporting global biosecurity efforts.

Mitochondrial DNA (mtDNA) methylation may be associated with mitochondrial damage; this study investigates their relationship in contrast-induced renal tubular epithelial cell (RTEC) injury. We stimulated HK-2 cells with iohexol to establish an in vitro model and analyzed the methylation level of mtDNA by bisulfite amplicon sequencing. The mitochondrial membrane potential, mitochondrial reactive oxygen species (mtROS), intracellular ROS, and changes in mitochondrial ultrastructure were evaluated as indicators of mitochondrial damage. Iohexol significantly inhibited cell viability and induced cell apoptosis, increasing both mtROS and intracellular ROS levels. Additionally, the methylation levels of mtDNA-encoded genes cytochrome c oxidase subunit I (COX I) (3.09%, *p < 0.05), cytochrome c oxidase subunit II (COX II) (4.51%, **p < 0.01), cytochrome c oxidase subunit III (COX III) (3.50%, **p < 0.01) and cytochrome B (CYTB)(4.66%, *p < 0.05) were increased, accompanied by enhanced transcription of both COX I and COX III. 5-Aza-dC, as a DNA methylation inhibitor, was dissolved in dimethyl sulfoxide (DMSO) vehicle to explore the role and mechanism of inhibiting mtDNA methylation in contrast-induced RTEC injury. HK-2 cells were further divided into four groups: vehicle control (DMSO alone), vehicle pretreated contrast - induced group (CI) (DMSO-CI), inhibitor control (5-Aza-dC), and inhibitor pretreated CI (5-Aza-dC-CI). Intriguingly, administration of 5-Aza-dC effectively attenuated mtDNA methylation, leading to improvements in these parameters and restoration of cell viability while reducing apoptosis. In conclusion, mtDNA methylation is involved in the mechanism of contrast-induced RTEC injury, potentially mediated by over-transcription of COX I and III, abnormal mtROS production, and subsequent mitochondrial damage and dysfunction. Inhibiting mtDNA methylation can provide protective effects against contrast - induced RTEC injury by reducing ROS (mtROS) production.

Ascorbate (ASC) is a key redox buffer in plant cells, whose antioxidant capacity depends on its balance with monodehydroascorbate (MDHA), its one-electron oxidation product. In the cytoplasm of Arabidopsis mesophyll cells, ASC is present at high concentrations and interacts with enzymes that oxidize it to MDHA, such as ascorbate peroxidases, as well as with enzymes that regenerate it, like NAD(P)H-dependent MDHA oxidoreductases (MDHAR) and glutathione-dependent dehydroascorbate reductases (DHAR). In vacuoles, ASC is found at lower concentrations and vacuoles lack these enzymes, but it can still undergo non-enzymatic oxidation by phenoxy radicals generated by class III peroxidases. It has been discovered that vacuoles isolated from Arabidopsis mesophyll cells contain an electron transport system that functionally connects the cytoplasmic and vacuolar ASC pools, acting as a transmembrane MDHA oxidoreductase dependent on Asc. Patch-clamp measurements have shown that electron currents across the tonoplast depend on the presence of ASC as an electron donor and MDHA or ferricyanide as electron acceptors on opposite sides of the membrane. These electron currents are catalyzed by cytochrome b561 isoform A (CYB561A), a tonoplast redox protein with ASC-binding sites in both the cytoplasm and the vacuole, electrically connected by two heme b groups. The recent functional characterization of other members of the cytochrome b561 family underscores how these proteins are essential for cellular redox balance and metabolism, facilitating electron transport across membranes and supporting processes such as iron homeostasis, stress defence, and cell wall modifications, highlighting their fundamental role in plant physiology.

Autophagy and apoplastic ascorbic acid (AsA) play important roles in plant drought tolerance. However, the trans-plasma membrane transport of AsA and its association with autophagy during drought stress are unclear. Here we report an AsA-induced autophagy pathway in plants, wherein plasma-membrane-located cytochrome b561 and DOMON domain (CYBDOM) proteins play positive roles. CYBDOM proteins from the resurrection plant Boea hygrometrica (BhDB) and Arabidopsis (AtDB1) can transport electrons across the plasma membrane using intracellular AsA as an electron donor and apoplastic monodehydroascorbic acid or Fe3+ as electron acceptors in Xenopus laevis oocytes. Increased apoplastic AsA, autophagy and drought tolerance are observed in BhDB- and AtDB1-overexpressing Arabidopsis compared with the wild type. CYBDOM proteins interact with respiratory burst oxidase homologue D (RbohD), which serves as the adaptor to bind autophagy related gene 8 protein (ATG8) and cargo protein for autophagic degradation. AsA increases AtDB1 protein level and its interaction with RbohD. Together, the AsA-activated CYBDOM-RbohD synergy to induce autophagy suggests a novel mechanism in plant drought and desiccation tolerance.