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1.
J Nutr Sci Vitaminol (Tokyo) ; 67(5): 339-350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34719620

RESUMO

5-Aminolevulinic acid (ALA) is a key intermediate of heme biosynthesis, which is an essential component of the respiratory chain. Therefore, nutrients that affect ALA biosynthesis eventually affect ATP production, which is the basis of mitochondrial function. Although the effects of various non-nutrient components that affect ALA after biosynthesis have been reported, there are few reports on the effects of dietary amino acids/protein on ALA formation and the effects of dietary vitamins that are involved in amino acid metabolism. In mitochondria, ALA is synthesized from succinyl-CoA and glycine by the pyridoxal phosphate-dependent enzyme ALA synthase [EC 2.3.1.37]. In this study, the effects of dietary amino acids/protein and vitamins on the amount of ALA synthesized were investigated using mice, rats, and cultured cells. Amounts of ALA in plasma and urine, and porphyrins in plasma increased with increasing protein intake. Vitamin B1 insufficiency did not affect ALA synthesis. Vitamin B6 insufficiency increased the amount of ALA synthesized, while niacin deficiency markedly reduced ALA synthesis. Thus, for heme synthesis, an essential biological substance for life, the amounts of amino acids, as well as the pathways metabolizing amino acids to glycine and succinyl-CoA are very important. Specifically, it is important that niacin is associated with the formation of glycine and succinyl-CoA from amino acids.


Assuntos
Ácido Aminolevulínico , Porfirinas , Aminoácidos , Animais , Glicina , Heme , Camundongos , Ratos
2.
PLoS One ; 16(10): e0256070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34653190

RESUMO

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Haemophilus influenzae/metabolismo , Heme/metabolismo , RNA de Cadeia Dupla/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Transporte/genética , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Otite Média/microbiologia , Otite Média/patologia , Conformação Proteica , Transporte Proteico/fisiologia , RNA de Cadeia Dupla/genética , Motivos de Ligação ao RNA/genética , Fatores de Virulência/metabolismo
3.
Redox Biol ; 47: 102170, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34688156

RESUMO

The role of heme oxygenase-1 in resisting oxidative stress and cell protection has always been a hot research topic. With the continuous deepening of research, in addition to directly regulating redox by catalyzing the degradation of heme, HO-1 protein also participates in the gene expression level in a great diversity of methods, thereby initiating cell defense. Particularly the non-canonical nuclear-localized HO-1 and HO-1 protein interactions play the role of a warrior against oxidative stress. Besides, HO-1 may be a promising marker for disease prediction and detection in many clinical trials. Especially for malignant diseases, there may be new advances in the treatment of HO-1 by regulating abnormal ROS and metabolic signaling. The purpose of this review is to systematically sort out and describe several aspects of research to facilitate further detailed mechanism research and clinical application promotion in the future.


Assuntos
Heme Oxigenase (Desciclizante) , Heme Oxigenase-1 , Heme/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Oxirredução , Estresse Oxidativo
4.
Molecules ; 26(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34641382

RESUMO

Protein glycation is an important protein post-translational modification and is one of the main pathogenesis of diabetic angiopathy. Other than glycated hemoglobin, the protein glycation of other globins such as myoglobin (Mb) is less studied. The protein glycation of human Mb with ribose has not been reported, and the glycation sites in the Mb remain unknown. This article reports that d-ribose undergoes rapid protein glycation of human myoglobin (HMb) at lysine residues (K34, K87, K56, and K147) on the protein surface, as identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Moreover, glycation by d-ribose at these sites slightly decreased the rate of the met heme (FeIII) in reaction with H2O2 to form a ferryl heme (FeIV=O). This study provides valuable insight into the protein glycation by d-ribose and provides a foundation for studying the structure and function of glycated heme proteins.


Assuntos
Compostos Férricos/química , Heme/química , Peróxido de Hidrogênio/química , Mioglobina/química , Ribose/química , Cromatografia Líquida , Glicosilação , Humanos , Espectrometria de Massas por Ionização por Electrospray
5.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639208

RESUMO

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Heme/química , Mutação , Peroxidase/química , Peroxidase/metabolismo , Proteínas de Bactérias/genética , Catálise , Domínio Catalítico , Corantes/química , Corantes/metabolismo , Estabilidade Enzimática , Heme/metabolismo , Concentração de Íons de Hidrogênio , Oxirredução , Peroxidase/genética , Conformação Proteica
6.
Redox Biol ; 46: 102125, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34517185

RESUMO

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.


Assuntos
Ferroquelatase , Proteínas Mitocondriais , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo
7.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576249

RESUMO

Human serum albumin (HSA) is the most abundant protein in plasma, contributing actively to oncotic pressure maintenance and fluid distribution between body compartments. HSA acts as the main carrier of fatty acids, recognizes metal ions, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays esterase, enolase, glucuronidase, and peroxidase (pseudo)-enzymatic activities. HSA-based catalysis is physiologically relevant, affecting the metabolism of endogenous and exogenous compounds including proteins, lipids, cholesterol, reactive oxygen species (ROS), and drugs. Catalytic properties of HSA are modulated by allosteric effectors, competitive inhibitors, chemical modifications, pathological conditions, and aging. HSA displays anti-oxidant properties and is critical for plasma detoxification from toxic agents and for pro-drugs activation. The enzymatic properties of HSA can be also exploited by chemical industries as a scaffold to produce libraries of catalysts with improved proficiency and stereoselectivity for water decontamination from poisonous agents and environmental contaminants, in the so called "green chemistry" field. Here, an overview of the intrinsic and metal dependent (pseudo-)enzymatic properties of HSA is reported to highlight the roles played by this multifaced protein.


Assuntos
Química Verde , Espécies Reativas de Oxigênio , Albumina Sérica Humana/química , Animais , Antioxidantes/química , Aspirina/química , Biomarcadores , Catálise , Frutose-Bifosfato Aldolase/metabolismo , Glucuronidase/química , Heme/química , Humanos , Íons , Ligantes , Peroxidação de Lipídeos , Conformação Molecular , Fosfopiruvato Hidratase/química , Ligação Proteica , Ratos
8.
Blood ; 138(13): 1092-1094, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34591096

Assuntos
Eritrócitos , Heme
9.
Anal Chem ; 93(37): 12758-12766, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34476936

RESUMO

Inspired by the key role of the coordination environment in the catalytic activity of enzymes, a rational design of the coordination structure of active sites at the atom scale is expected to develop high-performance enzyme-like catalysts. Here, we design a simple model system involving pentacoordinated and tetracoordinated Fe-N-C single-atom catalysts (named NG-Heme and G-Heme, respectively) to investigate structure-activity relationships. NG-Heme with axial ligand-engineered Fe sites exhibits superior enzyme-like activity to G-Heme, achieving the goal of vivid mimicking of the active sites of peroxidase. Experiments and theoretical studies reveal that the enhanced intrinsic catalytic activity originates from the "push effect" of the additional axial ligand, which can strengthen the interaction between the active site and the intermediate. Based on the outstanding catalytic activity, an NG-Heme-linked immunosorbent assay was constructed for colorimetric detection of carcinoembryonic antigen, exhibiting satisfactory sensitivity and feasibility in the analysis of clinical samples.


Assuntos
Heme , Peroxidase , Catálise , Imunoensaio , Ligantes
10.
Sensors (Basel) ; 21(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34502671

RESUMO

In recent years, the general and scientific interest in nutrition, digestion, and what role they play in our body has increased, and there is still much work to be carried out in the field of developing sensors and techniques that are capable of identifying and quantifying the chemical species involved in these processes. Iron deficiency is the most common and widespread nutritional disorder that mainly affects the health of children and women. Iron from the diet may be available as heme or organic iron, or as non-heme or inorganic iron. The absorption of non-heme iron requires its solubilization and reduction in the ferric state to ferrous that begins in the gastric acid environment, because iron in the ferric state is very poorly absorbable. There are chemical species with reducing capacity (antioxidants) that also have the ability to reduce iron, such as ascorbic acid. This paper aims to develop a sensor for measuring the release of encapsulated active compounds, in different media, based on dielectric properties measurement in the radio frequency range. An impedance sensor able to measure the release of microencapsulated active compounds was developed. The sensor was tested with calcium alginate beads encapsulating iron ions and ascorbic acid as active compounds. The prediction and measurement potential of this sensor was improved by developing a thermodynamic model that allows obtaining kinetic parameters that will allow suitable encapsulation design for subsequent release.


Assuntos
Ácido Ascórbico , Ferro , Antioxidantes , Criança , Dieta , Feminino , Compostos Férricos , Compostos Ferrosos , Heme , Humanos
11.
Int J Mol Sci ; 22(18)2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34576013

RESUMO

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from Klebsiella pneumoniae (KpDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Klebsiella pneumoniae/enzimologia , Peroxidase/metabolismo , Descoloração da Água , Aminoácidos/metabolismo , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Glicerol/metabolismo , Concentração de Íons de Hidrogênio
12.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556577

RESUMO

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos/metabolismo , Elétrons , Heme/metabolismo , Histidina/metabolismo , Metionina/metabolismo , Shewanella/metabolismo , Grupo dos Citocromos c/química , Citocromos/química , Transporte de Elétrons , Heme/química , Histidina/química , Metionina/química , Simulação de Dinâmica Molecular , Nanofios , Oxirredução
13.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576105

RESUMO

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the 'four-dimensional' protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the 'toolbox' of mass spectrometry researchers studying higher-order protein structure.


Assuntos
Peróxido de Hidrogênio/química , Ferro/química , Proteínas/química , Álcool Desidrogenase/química , Sítios de Ligação , Heme/química , Modelos Moleculares , Mioglobina/química , Oxirredução , Peptídeos/química , Conformação Proteica , Estabilidade Proteica , Proteína 1A de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/química
14.
Enzyme Microb Technol ; 150: 109883, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489036

RESUMO

Aromatic nitriles are important structural motifs that frequently existed in pharmaceutical drugs. Due to the convenient synthesis of aldoximes from aldehydes, the dehydration of aldoximes to corresponding nitriles by aldoxime dehydratases (Oxds) is considered as a safe and robust enzymatic production route. Although the Oxd genes are widely distributed in microbial kingdom, so far less than ten Oxds were expressed and further characterized. In this study, we found 26 predicted putative Oxd genes from the GenBank database using a genome mining strategy. The Oxd gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3). The amino acid sequence of OxdF1 shows high identities of 33∼85 % to other characterized Oxds, and contained a ferrous heme as the catalytic site. The optimum reaction pH and temperature of recombinant OxdF1 were 7.0 and 35 °C, respectively. OxdF1 was stable in pH 7.0 potassium phosphate buffer at 30 °C, and its half-life was approximately 3.8 h. OxdF1 can efficiently dehydrate aromatic and heterocyclic aldoximes to nitriles, such as 2-bromobenzaldoxime, 2-chloro-6-fluorobenzaldoxime, thiophene-2-carboxaldoxime, and pyridine-3-aldoxime. Therefore, the recombinant OxdF1 shows a potential application in the cyanide-free synthesis of aromatic nitriles.


Assuntos
Heme , Nitrilas , Pseudomonas putida/enzimologia , Hidroliases/genética , Oximas
15.
Int J Mol Sci ; 22(18)2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-34576282

RESUMO

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.


Assuntos
Citocromo P-450 CYP2A6/química , Citocromo P-450 CYP2A6/genética , Simulação de Dinâmica Molecular , Polimorfismo Genético , Sequência de Aminoácidos , Heme/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Proteínas Mutantes/química , Oxirredução , Estrutura Secundária de Proteína , Especificidade por Substrato
16.
Free Radic Biol Med ; 175: 95-107, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34478834

RESUMO

Hemopexin (Hpx) is a crucial defense protein against heme liberated from degraded hemoglobin during hemolysis. High heme stress creates an imbalance in Hpx bioavailability, favoring heme accumulation and downstream pathophysiological responses leading to cardiopulmonary disease progression in sickle cell disease (SCD) patients. Here, we evaluated a model of murine SCD, which was designed to accelerate red blood cell sickling, pulmonary hypertension, right ventricular dysfunction, and exercise intolerance by exposure of the mice to moderate hypobaric hypoxia. The sequence of pathophysiology in this model tracks with circulatory heme accumulation, lipid oxidation, extensive remodeling of the pulmonary vasculature, and fibrosis. We hypothesized that Hpx replacement for an extended period would improve exercise tolerance measured by critical speed as a clinically meaningful therapeutic endpoint. Further, we sought to define the effects of Hpx on upstream cardiopulmonary function, histopathology, and tissue oxidation. Our data shows that tri-weekly administrations of Hpx for three months dose-dependently reduced heme exposure and pulmonary hypertension while improving cardiac pressure-volume relationships and exercise tolerance. Furthermore, Hpx administration dose-dependently attenuated pulmonary fibrosis and oxidative modifications in the lung and myocardium of the right ventricle. Observations in our SCD murine model are consistent with pulmonary vascular and right ventricular pathology at autopsy in SCD patients having suffered from severe pulmonary hypertension, right ventricular dysfunction, and sudden cardiac death. This study provides a translational evaluation supported by a rigorous outcome analysis demonstrating therapeutic proof-of-concept for Hpx replacement in SCD.


Assuntos
Anemia Falciforme , Hemopexina , Anemia Falciforme/tratamento farmacológico , Animais , Heme , Hemoglobinas , Hemólise , Humanos , Camundongos
17.
Artigo em Russo | MEDLINE | ID: mdl-34460162

RESUMO

OBJECTIVE: To compare the antioxidant effects of cortexin, cerebrolysin and actovegin in rats with chronic brain ischemia. MATERIAL AND METHODS: Chronic brain ischemia was modeled in male rats by 50% stenosis of the common carotid arteries. Forty days after surgery, the animals received 2 ten-day courses of therapy, separated by a break of 10 days. Placebo, cortexin (0.3, 1 and 3 mg/kg), cerebrolysin (0.8, 2.5 and 7.5 ml/kg) and actovegin (5 ml/kg) were administered to animals as treatment. The concentration of malondialdehyde (MDA) in the homogenates was determined by the reaction with thiobarbituric acid, the concentration of reduced glutathione was determined by the reduction reaction of 5.5-dithiobis- (2-nitrobenzoic acid); determination of catalase activity, as well as the content of lactate and pyruvate, by commercially available reagent kits. The activity of superoxide dismutase (SOD) was determined by the photometric method based on an assessment of the degree of inhibition of the epinephrine oxidation reaction. All reactions were carried out in triplicates. RESULTS: Modeling of chronic brain ischemia led to the statistically significant decrease in the content of lactate and pyruvate (p<0.001, when compared with the control group), which was not accompanied by a significant decrease in their ratio (p>0.05), as well as to the decrease in SOD, catalase activity, restored glutathione and increase in MDA concentrations. Compared with the control group, in the groups that received cortexin at a dose of 3 mg/kg/day, cerebrolysin at a dose of 7.5 ml/kg/day and actovegin at a dose of 5 ml/kg/day, there were an increase in the content of lactate and pyruvate (without a significant change in their ratio), restoration of glutathione levels and the activity of SOD and, to a lesser extent, catalase, combined with a decrease in the concentration of MDA. CONCLUSION: Course administration of cortexin (3 mg/kg), cerebrolysin (7.5 ml/kg) and, to a lesser extent, actovegin (5 ml/kg) has a positive effect on the state of the antioxidant system of the brain in rats with chronic brain ischemia.


Assuntos
Antioxidantes , Isquemia Encefálica , Aminoácidos , Animais , Isquemia Encefálica/tratamento farmacológico , Heme/análogos & derivados , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Ratos , Ratos Wistar
18.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443440

RESUMO

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN- ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.


Assuntos
Metaloproteínas/química , Análise Espectral Raman , Heme/química , Proteínas Ferro-Enxofre/química , Oxirredução , Espectrofotometria Infravermelho
19.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445350

RESUMO

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.


Assuntos
Hemorragia Cerebral/patologia , Plexo Corióideo/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/patologia , Proteína C-Reativa/líquido cefalorraquidiano , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Hemorragia Cerebral/complicações , Hemorragia Cerebral/congênito , Hemorragia Cerebral/metabolismo , Plexo Corióideo/patologia , Estudos de Coortes , Citocinas/líquido cefalorraquidiano , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Heme/metabolismo , Hemoglobinas/metabolismo , Humanos , Hungria , Recém-Nascido , Recém-Nascido Prematuro , Molécula 1 de Adesão Intercelular/líquido cefalorraquidiano , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Síndrome de Resposta Inflamatória Sistêmica/congênito , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Molécula 1 de Adesão de Célula Vascular/líquido cefalorraquidiano , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
Biomed Res Int ; 2021: 6679076, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337043

RESUMO

Linezolid can cause serious haematological toxicity, such as thrombocytopenia and aneamia. Heme, composed of iron and porphyrin, is an important component of hemoglobin. In order to investigate the relationship between the concentration of linezolid and heme in the plasma of infected patients, a UPLC-MS/MS method that can determine the concentrations of linezolid and heme simultaneously was developed and validated. A total of 96 healthy subjects and 81 infected patients, who received blood routine blood tests, were included and determined by the UPLC-MS/MS method. The results showed that the concentration of linezolid was 5.08 ± 3.46 µg/mL in infected patients who were treated with linezolid. The heme in healthy subjects was 7.05 ± 8.68 µg/mL, and it was significantly decreased to 0.88 ± 0.79 µg/mL in infected patients (P < 0.01). Spearman correlation analysis showed that linezolid had a high negative correlation with platelet (PLT) (R = -0.309). Heme had a high positive correlation with hemoglobin (Hb) (R = 0.249) in healthy subjects and infected patients. The ROC analysis showed that heme had diagnostic value to distinguish low Hb (110 g/L). In conclusion, there was a positive correlation between heme and Hb, and this correlation was also observed in infected patients. A high concentration of linezolid was inclined to decrease PLT. Monitoring of heme and linezolid helps in the early diagnose of low Hb and PLT.


Assuntos
Heme/análise , Infecções/sangue , Linezolida/sangue , Espectrometria de Massas em Tandem , Adulto , Plaquetas/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Hematócrito , Testes Hematológicos , Humanos , Rim/fisiopatologia , Fígado/fisiopatologia , Masculino , Pessoa de Meia-Idade , Curva ROC
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