RESUMO
Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of â¼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.
Assuntos
Echinococcus granulosus , Proteínas de Helminto , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Animais , Cães , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/química , Bovinos , Sequência de Aminoácidos , Tripsina/química , Tripsina/metabolismoRESUMO
OBJECTIVE: This study aimed to assess the efficacy and tolerability of stereotactic body radiation therapy (SBRT) for the treatment of liver metastases. METHODS: Patients with up to 5 liver metastases were enrolled in this prospective multicenter study and underwent SBRT. Efficacy outcomes included in-field local control (LC), progression-free survival (PFS), and overall survival (OS). Acute and late toxicities were evaluated using CTCAE v.4.0. RESULTS: A total of 52 patients with 105 liver metastases were treated between 2015 and 2018. The most common primary tumor was colorectal cancer (72% of cases). Liver metastases were synchronous with the primary tumor diagnosis in 24 patients (46.2%), and 21 patients (40.4%) presented with other extrahepatic oligometastases. All patients underwent intensity-modulated radiation therapy (IMRT)/volumetric-modulated arc therapy (VMAT) with image-guided radiation therapy (IGRT) and respiratory gating, and a minimum biologically effective dose (BED10Gy) of 100 Gy was delivered to all lesions. With a median follow-up of 23.1 months (range: 13.4-30.9 months) since liver SBRT, the median actuarial local progression-free survival (local-PFS) was not reached. The actuarial in-field LC rates were 84.9% and 78.4% at 24 and 48 months, respectively. The median actuarial liver-PFS and distant-PFS were 11 and 10.8 months, respectively. The actuarial median overall survival (OS) was 27.7 months from SBRT and 52.5 months from metastases diagnosis. Patients with lesion diameter ≤ 5 cm had significantly better median liver-PFS (p = 0.006) and OS (p = 0.018). No acute or late toxicities of grade ≥ 3 were observed. CONCLUSIONS: This prospective multicenter study confirms that liver SBRT is an effective alternative for the treatment of liver metastases, demonstrating high rates of local control and survival while maintaining a low toxicity profile.
Assuntos
Neoplasias Hepáticas , Radiocirurgia , Radioterapia de Intensidade Modulada , Humanos , Radiocirurgia/métodos , Radiocirurgia/efeitos adversos , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/mortalidade , Masculino , Estudos Prospectivos , Feminino , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto , Radioterapia de Intensidade Modulada/métodos , Intervalo Livre de Progressão , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Neoplasias Colorretais/mortalidade , Radioterapia Guiada por Imagem , Taxa de SobrevidaRESUMO
In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.
Assuntos
Microbiota , Poluentes Químicos da Água , Rios/química , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , Cuba , Catalase , Peptídeo Hidrolases , Lipase , Preparações Farmacêuticas , Monitoramento Ambiental/métodosRESUMO
BACKGROUND: Preoperative radiation therapy following by limb-sparing or conservative surgery is a standard approach for limb and trunk STS. Data supporting hypofractionated radiotherapy schedules are scarce albeit biological sensitivity of STS to radiation would justify it. We sought to evaluate the impact of moderate hypofractionation on pathologic response and its influence on oncologic outcomes. MATERIAL AND METHODS: From October 2018 to January 2023, 18 patients with limb or trunk STS underwent preoperative radiotherapy at a median dose of 52.5 Gy (range 49.5-60 Gy) in 15 fractions of 3.5 Gy (3.3-4 Gy) with or without neoadjuvant chemotherapy. A favorable pathologic response (fPR) was considered as ≥ 90% tumor necrosis on specimen examination. RESULTS: All patients completed planned preoperative radiotherapy. Eleven patients (61.1%) achieved a fPR, and 7 patients (36.8%) a complete pathologic response with total disappearance of tumor cells. Nine patients (47%) developed grade 1-2 acute skin toxicity, and 7 patients (38.8%) had wound complications on follow-up. With a median follow-up of 14 months (range 1-40), no cases of local relapse were observed, and actuarial 3-year overall survival (OS) and distant metastases-free survival (DMFS) are 87% and 76.4%, respectively. In the univariate analysis, the presence of a favorable pathologic response (fPR) was associated with improved 3-year OS (100% vs. 56.03%, p = 0.058) and 3-year DMFS (86.91% vs. 31.46%, p = 0.002). Moreover, both complete or partial RECIST response and radiological stabilization of the tumor lesion showed a significant association with higher rates of 3-year distant metastasis-free survival (DMFS) (83% vs. 83% vs. 56%, p < 0.001) and 3-year overall survival (OS) (100% vs. 80% vs. 0, p = 0.002). CONCLUSIONS: Preoperative moderate hypofractionated radiation treatment for STS is feasible and well tolerated and associates encouraging rates of pathologic response that could have a favorable impact on final outcomes.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Hipofracionamento da Dose de Radiação , Recidiva Local de Neoplasia/patologia , Extremidades/patologia , Sarcoma/patologia , Terapia Neoadjuvante , Neoplasias de Tecidos Moles/radioterapia , Neoplasias de Tecidos Moles/patologia , Resultado do Tratamento , Estudos RetrospectivosRESUMO
OBJECTIVE: To assess the clinical outcomes of patients with spine metastases treated with SBRT at our institution. MATERIALS AND METHODS: Patients with spine metastases treated with SBRT (1 fraction/18 Gy or 5 fractions/7 Gy) during the last 12 years have been analyzed. All patients were simulated supine in a vacuum cushion or with a shoulder mask. CT scans and MRI image registration were performed. Contouring was based on International Spine-Radiosurgery-Consortium-Consensus-Guidelines. Highly conformal-techniques (IMRT/VMAT) were used for treatment planning. Intra and interfraction (CBCT or X-Ray-ExacTrac) verification were mandatory. RESULTS: From February 2010 to January 2022, 129 patients with spinal metastases were treated with SBRT [1 fraction/18 Gy (75%) or 5 fractions/7 Gy] (25%). For patients with painful metastases (74/129:57%), 100% experienced an improvement in pain after SBRT. With a median follow-up of 14.2 months (average 22.9; range 0.5-140) 6 patients (4.6%) experienced local relapse. Local progression-free survival was different, considering metastases's location (p < 0.04). The 1, 2 and 3 years overall survival (OS) were 91.2%, 85.1% and 83.2%, respectively. Overall survival was significantly better for patients with spine metastases of breast and prostate cancers compared to other tumors (p < 0.05) and significantly worse when visceral metastases were present (p < 0.05), when patients were metastatic de novo (p < 0.05), and in those patients receiving single fraction SBRT (p: 0.01). CONCLUSIONS: According to our experience, SBRT for patients with spinal metastases was effective in terms of local control and useful to reach pain relief. Regarding the intent of the treatment, an adequate selection of patients is essential to propose this ablative approach.
Assuntos
Radiocirurgia , Neoplasias da Coluna Vertebral , Masculino , Humanos , Radiocirurgia/métodos , Neoplasias da Coluna Vertebral/radioterapia , Neoplasias da Coluna Vertebral/cirurgia , Neoplasias da Coluna Vertebral/patologia , Recidiva Local de Neoplasia/etiologia , Mama/patologia , Dor/etiologia , Estudos RetrospectivosRESUMO
DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: ⢠An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. ⢠The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. ⢠rDyP-AU10 showed high activity at low temperatures. ⢠rDyP-AU10 is potentially useful for biotechnological applications.
Assuntos
Corantes , Peroxidase , Peroxidase/metabolismo , Corantes/metabolismo , Escherichia coli/genética , Regiões Antárticas , Peróxido de Hidrogênio , Peroxidases/metabolismoRESUMO
Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.
Assuntos
Bacillus subtilis , Transdução de Sinais , Histidina Quinase/metabolismo , Bacillus subtilis/genética , Fosforilação , Monoéster Fosfórico Hidrolases , Proteínas de Bactérias/metabolismoRESUMO
Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.
RESUMO
The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.
Assuntos
Ácido Aspártico Proteases , Vírus da Leucemia Bovina , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Protease de HIV/metabolismo , Cinética , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Introducción: El proceso de adherencia es fundamental en el desarrollo de la mayoría de las infecciones ocasionadas por Escherichia coli. Para los patotipos de esta especie se describen tres patrones de adherencia diferentes: adherencia localizada, adherencia difusa y adherencia agregativa, los cuales se relacionan con los procesos patogénicos específicos que ocasiona en la clínica. Sin embargo, son pocos los estudios en relación con los fenotipos de adherencia in vitro de E. coli aisladas del ambiente. Objetivo: Determinar los fenotipos de adherencia de cepas de E. coli aisladas de ecosistemas dulceacuícolas de La Habana. Métodos: Se analizaron 108 cepas de E. coli aisladas de los ríos Almendares, Quibú y Luyanó de La Habana. Se determinó el patrón de adherencia mediante ensayos de adherencia en cultivo celular de la línea HEp-2 así como el serotipo de cada cepa. Resultados: El 25 por ciento de las cepas de E. coli aisladas fueron adherentes y el 75 por ciento fueron no adherentes. Veintidós cepas mostraron el típico patrón de adherencia difusa y cinco cepas mostraron una adherencia agregativa. Se encontraron cepas de los dos patrones de adherencia en los tres ríos evaluados. Las cepas presentaron 24 serotipos diferentes. Conclusiones: Se demostró que las cepas de E. coli ambientales circulantes en estos ecosistemas presentan características adherentes, cuya patogenicidad implica un riesgo potencial para la salud humana, especialmente en edades pediátricas(AU)
Introduction: Adherence is crucial to the development of most Escherichia coli infections. Three different adherence patterns have been described for pathotypes of this species: localized, diffuse and aggregative adherence, based on the specific clinical pathogenic processes they bring about. However, few studies have been conducted about in vitro adherence phenotypes of E. coli isolated from the environment. Objective: Determine the adherence phenotypes of E. coli strains isolated from freshwater ecosystems in Havana. Methods: An analysis was conducted of 108 E. coli strains isolated from the rivers Almendares, Quibú and Luyanó in Havana. Determination was made of the adherence pattern by adherence assays in HEp-2 cell line cultures, as well as of the serotype for each strain. Results: Of the E. coli strains isolated, 25 percent were adherent and 75 percent were not. Twenty-two strains displayed the typical diffusely adherent pattern and five displayed aggregative adherence. Strains exhibiting the two adherence patterns were found in the three rivers evaluated. The strains contained 24 different serotypes. Conclusions: The environmental E. coli strains circulating in these ecosystems were found to have adherent characteristics whose pathogenicity implies a potential risk to human health, particularly in childhood(AU)
Assuntos
Humanos , Masculino , Feminino , Ecossistema , Infecções por Escherichia coli , Água DoceRESUMO
Introducción: Las cepas de Escherichia coli productoras de β-lactamasas de espectro extendido son patógenos multirresistentes y una de las bacterias que más contribuyen con la resistencia antibiótica bacteriana en la clínica. Sin embargo, se aíslan cada vez con más frecuencia de ambientes naturales, tales como los ecosistemas acuáticos en los cuales se emplea como un indicador de contaminación fecal. Objetivo: Evaluar la susceptibilidad a los antibióticos y la producción de enzimas ß-lactamasas de espectro extendido de aislados de Escherichia coli procedentes de ecosistemas dulceacuícolas de La Habana. Métodos: Se analizaron 43 aislados de E. coli provenientes de los ríos Almendares, Quibú y Luyanó de La Habana. Se determinó la susceptibilidad a 18 antibióticos y la producción fenotípica de ß-lactamasas de espectro extendido según las normas del Instituto de Estándares para el Laboratorio Clínico. La detección molecular de las enzimas se realizó mediante reacción en cadena de la polimerasa. Se calculó el índice de multirresistencia a los antibióticos y los patrones de resistencia de cada aislado de E. coli- ß-lactamasas de espectro extendido. Resultados: El 65 por ciento de los aislados de E. coli fueron resistentes al menos a un antibiótico y el 35 por ciento fueron sensibles a todos los antibióticos. El fenotipo ß-lactamasas de espectro extendido fue detectado en siete aislados; de estos, cuatro fueron portadores del gen bla CTX-M-1 y tres presentaron bla TEM. El 37 por ciento de los aislados de E. coli mostraron valores de índices de multirresistencia a los antibióticos menores que 0,22; el 16 por ciento de 0,22; el 9,3 por ciento mayor que 0,5; y el 5 por ciento mayor que 0,7. Los aislados de E. coli-BLEE mostraron corresistencia a las familias de las tetraciclinas, quinolonas, aminoglucósidos y macrólidos. Conclusiones: La presencia de aislados ambientales multirresistentes de E. coli productores de ß-lactamasas de espectro extendido en ecosistemas dulceacuícolas de La Habana destaca la necesidad de implementar estrategias de control para prevenir la diseminación de estos aislados en los ambientes naturales(AU)
Introduction: Extended-spectrum β-lactamase-producing Escherichia coli strains are multiresistant pathogens and one of the bacteria contributing most greatly to bacterial antibiotic resistance in clinical practice. However, they are increasingly isolated from natural environments, such as aquatic ecosystems, where they are used as fecal pollution indicators. Objective: Evaluate antibiotic susceptibility and extended-spectrum ß-lactamase enzyme production in Escherichia coli isolates from freshwater ecosystems in Havana. Methods: An analysis was conducted of 43 E. coli isolates from the rivers Almendares, Quibú and Luyanó in Havana. Determination was made of susceptibility to 18 antibiotics and phenotypic production of extended-spectrum ß-lactamases according to standards from the Clinical and Laboratory Standards Institute. Molecular detection of the enzymes was performed by polymerase chain reaction. Estimation was carried out of the antibiotic multiresistance index and the resistance patterns of each extended-spectrum E. coli ß-lactamase isolate. Results: Of the E. coli isolates studied, 65 percent were resistant to at least one antibiotic, whereas 35 percent were sensitive to all antibiotics. The extended-spectrum ß-lactamase phenotype was detected in seven isolates, of which four were carriers of the gene bla CTX-M-1 and three contained bla TEM. 37 percent of the E. coli isolates displayed antibiotic multiresistance index values below 0.22, 16 percent of 0.22, 9.3 percent above 0.5 and 5 percent above 0.7. ESBL E. coli isolates displayed co-resistance to the families tetracyclines, quinolones, aminoglycosides and macrolides. Conclusions: The presence of multiresistant extended-spectrum ß-lactamase-producing environmental E. coli isolates in Havana freshwater ecosystems highlights the need to implement control strategies aimed at preventing the spread of these isolates in natural environments(AU)
Assuntos
Humanos , Resistência Microbiana a Medicamentos , Reação em Cadeia da Polimerase , Ecossistema , Suscetibilidade a Doenças , Poluição Ambiental , Escherichia coli , Água Doce , Padrões de Referência , Indicadores de ContaminaçãoRESUMO
Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.
Assuntos
Antioxidantes/química , Cisteína/genética , Albumina Sérica Humana/genética , Compostos de Sulfidrila/química , Cromatografia por Troca Iônica , Cisteína/química , Expressão Gênica/genética , Humanos , Domínios Proteicos/genética , Multimerização Proteica , Saccharomycetales/genética , Albumina Sérica Humana/químicaRESUMO
Persulfides (RSSH/RSS-) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS-), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS- presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, ßnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.
Assuntos
Dissulfetos/química , Glutationa/análogos & derivados , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Dissulfetos/análise , Dissulfetos/metabolismo , Glutationa/análise , Glutationa/química , Glutationa/metabolismo , Peróxido de Hidrogênio/química , Sulfeto de Hidrogênio/química , Sulfeto de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ácido Peroxinitroso/química , Teoria Quântica , Espectrometria de Massas em Tandem , TermodinâmicaRESUMO
Persulfides (RSSH/RSS-) can be formed in protein and non-protein thiols (RSH) through several different pathways, some of which are dependent on hydrogen sulfide (H2S/HS-). In addition to their roles in biosynthetic processes, persulfides are possible transducers of physiological effects of H2S through the modification of critical cysteines. Persulfides have a very rich biological chemistry that is currently under investigation. They are more nucleophilic and acidic than thiols and, unlike thiols, they can also be electrophilic. They are especially good one-electron reductants. Methods to detect their formation are under continuous development. In this minireview we describe the pathways of formation of persulfides, their biochemical properties and the techniques available for their detection, and we discuss the possible implications of their formation in biological systems.
Assuntos
Proteínas/metabolismo , Sulfetos/metabolismo , Animais , Humanos , Sulfeto de Hidrogênio/química , Proteínas/análise , Proteínas/química , Proteômica , Compostos de Sulfidrila/química , Sulfetos/análise , Sulfetos/químicaRESUMO
The free radical nitric oxide (NOâ¢) exerts biological effects through the direct and reversible interaction with specific targets (e.g. soluble guanylate cyclase) or through the generation of secondary species, many of which can oxidize, nitrosate or nitrate biomolecules. The NOâ¢-derived reactive species are typically short-lived, and their preferential fates depend on kinetic and compartmentalization aspects. Their detection and quantification are technically challenging. In general, the strategies employed are based either on the detection of relatively stable end products or on the use of synthetic probes, and they are not always selective for a particular species. In this study, we describe the biologically relevant characteristics of the reactive species formed downstream from NOâ¢, and we discuss the approaches currently available for the analysis of NOâ¢, nitrogen dioxide (NO2â¢), dinitrogen trioxide (N2O3), nitroxyl (HNO), and peroxynitrite (ONOO-/ONOOH), as well as peroxynitrite-derived hydroxyl (HOâ¢) and carbonate anion (CO3â¢-) radicals. We also discuss the biological origins of and analytical tools for detecting nitrite (NO2-), nitrate (NO3-), nitrosyl-metal complexes, S-nitrosothiols, and 3-nitrotyrosine. Moreover, we highlight state-of-the-art methods, alert readers to caveats of widely used techniques, and encourage retirement of approaches that have been supplanted by more reliable and selective tools for detecting and measuring NOâ¢-derived oxidants. We emphasize that the use of appropriate analytical methods needs to be strongly grounded in a chemical and biochemical understanding of the species and mechanistic pathways involved.
Assuntos
Radicais Livres/química , Óxido Nítrico/química , Oxidantes/química , Biologia de Sistemas , Radicais Livres/metabolismo , Humanos , Radical Hidroxila/química , Nitratos/química , Óxido Nítrico/genética , Oxirredução , Ácido Peroxinitroso/química , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/genéticaRESUMO
Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.
Assuntos
Cisteína/análogos & derivados , Dissulfetos/metabolismo , Mycobacterium tuberculosis/metabolismo , Peroxirredoxinas/metabolismo , Catálise , Domínio Catalítico , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Peróxido de Hidrogênio/química , Sulfeto de Hidrogênio/metabolismo , Cinética , Oxirredução , Especificidade por Substrato , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , SulfetosRESUMO
Carbonate radicals (CO3-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3- was followed by visible spectrophotometry. Solutions containing PGR (5-200 µM), SOD-1 (0.3-3 µM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3- release of 24.6⯱â¯4.3 µM min-1 for 3 µM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2⯱â¯0.1 µM min-1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3- release from bSOD-1 and hSOD-1 peroxidase activity.
Assuntos
Bicarbonatos/química , Clareadores/química , Carbonatos/química , Radicais Livres/química , Pirogalol/análogos & derivados , Superóxido Dismutase-1/química , Bicarbonatos/metabolismo , Carbonatos/metabolismo , Radicais Livres/metabolismo , Oxirredução , Pirogalol/química , Análise Espectral , Superóxido Dismutase-1/metabolismoRESUMO
Hydrogen sulfide (H2S/HSâ») can be formed in mammalian tissues and exert physiological effects. It can react with metal centers and oxidized thiol products such as disulfides (RSSR) and sulfenic acids (RSOH). Reactions with oxidized thiol products form persulfides (RSSH/RSSâ»). Persulfides have been proposed to transduce the signaling effects of H2S through the modification of critical cysteines. They are more nucleophilic and acidic than thiols and, contrary to thiols, also possess electrophilic character. In this review, we summarize the biochemistry of hydrogen sulfide and persulfides, focusing on redox aspects. We describe biologically relevant one- and two-electron oxidants and their reactions with H2S and persulfides, as well as the fates of the oxidation products. The biological implications are discussed.
RESUMO
Los materiales poliméricos han sido ampliamente investigados para aplicaciones biomédicas, teniendo especial relevancia cuando se encuentran en forma de micro- y nano-partículas. Últimamente se ha ampliado su campo de aplicación al ser conjugados con péptidos y ácidos nucleicos, por lo tanto, el interés en el estudio de este tipo de materiales, así como también en la formulación de nanoestructuras funcionalizadas como materiales, dispositivos y vehículos de transporte de agentes terapéuticos ha aumentado. Las recientes investigaciones en nanosistemas se inspiran en fenómenos naturales que estimulan la integración de señales moleculares y la mimetización de procesos a nivel celular, de tejidos y órganos. Tecnológicamente, la capacidad de obtener nanoestructuras esféricas mediante la combinación de materiales que presenten propiedades distintas a las que ningún otro material individual posee por sí solo, es lo que hace que las nanocápsulas sean particularmente atractivas. Las potenciales ventajas de los sistemas de nanopartículas de tipo polimérico se destacan a lo largo de cada parte de este artículo de revisión. El presente artículo aborda los aspectos más relevantes sobre la estructura, composición y algunos métodos de elaboración de los sistemas nanoparticulados. Además, expone algunos de los trabajos más recientes, centrados en sistemas de nanopartículas basados en polímeros dirigidos a la administración de agentes, publicados en artículos especializados de investigación y revisiones durante los últimos años.
Polymeric materials have been extensively investigated for biomedical applications including micro- and nanoparticles. Modern advances have broadened horizons for application with peptides and nucleic acids. Therefore, interests increased in the formulation of materials, devices and vehicles for transporting therapeutic agents in functionalized nanostructures. Recent nano-systems are inspired by natural phenomena that stimulate the integration of molecular signals and the mimicking of natural cellular processes, at tissue and organ levels. Technologically, the ability to obtain spherical nanostructures, which combine different properties, that no other single material possesses on its own, makes nanocapsules particularly attractive. Potential advantages over polymer nanoparticulate systems are highlighted throughout each part of this review article. Here, we address the most relevant aspects of structure, composition and methods of formulation of nanoparticulate systems. In addition, we outline some of the more recent works focusing on nanosized preparations, based on agent-directed polymers, found in specialized research articles that have emerged in the recent years.
Assuntos
Polímeros/química , Nanopartículas/química , Sistemas de Liberação de Medicamentos , Engenharia Tecidual , Pontos Quânticos , Nanocápsulas/química , Nanosferas/químicaRESUMO
Nanotecnología es la ciencia que involucra la síntesis de materiales en escala entre 1-100 nm (nanomateriales) es aplicable en diferentes áreas tales como medio ambiente, electrónica, alimentos, energía, entre otros. Los campos que serán relevantes dentro de esta revisión y explicados en detalle son la nanomedicina y la nano-odontología. Actualmente, en estas áreas los tres principales temas en desarrollo son específicamente en el sub-área de la nanobiotecnología y corresponden a: sensorización (biosensores/biodetección), diagnóstico (biomarcadores/bioimagen) y transportes de genes, proteínas o fármacos (sistemas de intercambio controlado en blancos sistémicos versus localizados). También se han presentado avances en bioaplicaciones como modelamientos de membranas, marcaje celular, entrega de agentes a blancos específicos, estrategias para prevención de enfermedades, ingeniería de tejidos, regeneración de órganos, estrategias de inmunoensayos y nano-oncología. Este artículo de revisión pretende abordar algunos de los aportes más relevantes, que tienen algunos de los trabajos recientes, sobre los sistemas de nanopartículas, principalmente aquellos dirigidos a terapias en áreas como diabetes, nano-oncología, terapia de fármacos y genes, mediante la técnica layer-by-layer y autoensamblado, muy utilizados también en ingeniería de tejidos y regeneración tisular, junto a un breve resumen de los avances que existen en el campo de la nano-odontología.
Nanotechnology is the science that involves the synthesis of materials in scale between 1-100 nm (nanomaterials) and is applicable in different areas such as environment, electronics, food, energy, among others. The fields that will be relevant within this review and explained in detail are nanomedicine and nano-dentistry. Currently, in these areas, the three main topics under development are specifically in the sub-area of nanobiotechnology and correspond to: sensorization (biosensors / biosensing), diagnostics (biomarkers / bioimaging) and transport of genes, proteins or drugs (exchange systems) controlled in systemic versus localized targets). Advances have also been presented in bioapplications such as membrane modeling, cell marking, delivery of agents to specific targets, strategies for disease prevention, tissue engineering, organ regeneration, immunoassay strategies and nano-oncology. This review article aims to address some of the most relevant contributions, some of the recent work, on nanoparticle systems, mainly those aimed at therapies in areas such as diabetes, nanooncology, drug and gene therapy, through the layer-by-layer and self-assembled technique, also widely used in tissue engineering and tissue regeneration, together with a brief summary of the advances that exist in the field of nano-dentistry.