RESUMO
The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Apoptose , Leishmania tropica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antiprotozoários/química , Venenos de Abelha/química , Cálcio/análise , Cálcio/metabolismo , Linhagem Celular , Dicroísmo Circular , Fragmentação do DNA , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Fluorometria , Humanos , Leishmania tropica/ultraestrutura , Leishmaniose Visceral/parasitologia , Macrófagos Peritoneais , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mitocôndrias/química , Tamanho Mitocondrial , Espécies Reativas de Oxigênio/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxidos/isolamento & purificaçãoRESUMO
The superoxide anion (O2â¢-) is an important reactive oxygen species (ROS) in the brain system, which has been associated with the development of many neurological diseases, including Alzheimer's disease (AD). Herein, we introduced a carbon fiber microelectrode (CFME) based in vivo technique for specific and sensitive monitoring of the O2â¢- radical in the living brains of both normal and AD model rats. Compared with other reported superoxide dismutase (SOD) electrochemical biosensors, the microsensor presented in our work was featured in the coating of a functionalized ionic liquid polymer (PIL) onto PB nanoparticles (PBNPs) and carbon nanotubes (CNT). It was demonstrated that the cationic and carboxyl-rich PILs provided abundant interaction sites with SOD to prevent enzyme leakage from sensor, which was beneficial for the enhancement of sensitivity. Additionally, CCK-8 assay and autoxidation of pyrogallol tests showed that MCF-7â¯cells maintained a high viability after incubated with PIL and most of the SOD bioactivity was retained in the presence of PIL, which implied the PIL itself possessed an excellent biocompatibility. These properties allow the sensor to track the fluctuation of O2â¢- levels in vivo between normal and AD rats. This is the first report on application of functionalized PIL to reveal the O2â¢- related pathological process of AD.
Assuntos
Doença de Alzheimer/metabolismo , Técnicas Biossensoriais , Encéfalo/metabolismo , Superóxidos/isolamento & purificação , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/patologia , Animais , Fibra de Carbono/química , Humanos , Líquidos Iônicos/química , Íons , Células MCF-7 , Microeletrodos , Nanopartículas/química , Nanotubos de Carbono/química , Polímeros/química , Ratos , Espécies Reativas de Oxigênio/química , Superóxido Dismutase/química , Superóxidos/químicaRESUMO
Monitoring superoxide anions in living cells have attracted much academic and biomedical interest due to their important role in metabolic processes. Herein, we confined ultra-small nano-Mn3(PO4)2 in chitosan and designed a unique puffy woven sphere consisted by nanowires. Further constructed an effective in situ detection chip using the as-synthesized nano-Mn3(PO4)2-chitosan for electrochemical sensing of superoxide anions from murine breast tumor cells (4T1). The excellent biocompatibility of chitosan and large size of the Mn3(PO4)2-chitosan spheres greatly reduced the damage and toxicity of the detection interface to the living cells, while the ultra-small nano-Mn3(PO4)2 in chitosan could effectively catalyze the superoxide anions released from cells. The nano-Mn3(PO4)2-chitosan-based sensor exhibited high sensitivity (1.6 µA µM-1), low detection limit (9.4â¯nM at S/Nâ¯=â¯3) and good selectivity for O2â¢-. After cell culture on the surface of nano-Mn3(PO4)2-chitosan based electrode. As a miniature analytical and sensing platform, results further suggest that the prepared chip offers a more sensitive detective superoxide anions (O2â¢-) released from 4T1 cell lines than traditional electron paramagnetic resonance (EPR) analysis.
Assuntos
Técnicas Biossensoriais , Quitosana/química , Técnicas Eletroquímicas , Superóxidos/isolamento & purificação , Enzimas Imobilizadas/química , Humanos , Manganês/química , Nanopartículas Metálicas/química , Superóxidos/químicaRESUMO
Here we present a new method to fabricate enzyme-mimic metal-free catalysts for electrochemical detection of superoxide anion (O2â¢-) by introducing phosphate groups into graphene-based foam. Through a template-free hydrothermal process, graphene oxide (GO) was treated with different amount of phytic acid (PA) to obtain 3D porous graphene-based foam (PAGF). Characterizations demonstrate that phosphate groups were successfully modified on the surface and inter layer structure of PAGF materials and the defects and disorder degree of PAGF could be controlled by adjusting the addition of PA precursors. Meanwhile, the synthesized PAGF was successfully immobilized on screen printed carbon electrodes (SPCEs) and employed in O2â¢- detection. With PA treated on graphene structure, the resulted PAGF/SPCEs exhibit distinct characteristic redox peaks, showing enzyme-mimic catalytic activity toward O2â¢- dismutation. Also, the amount of modified phosphate groups has caused a considerable variety on the performance of PAGF-based electrodes. Apart from high sensitivity, wide liner range, low detection limit, good selectivity and long-term stability, our sensors also present satisfying performance in the real-time monitoring of drug-induced O2â¢- released from Hela cells. The reliability of the biological measurement was further demonstrated via electron paramagnetic resonance (EPR) to characterize the released O2â¢- from stimulated cells by using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) to trap the transient O2â¢-. The above results indicate that our established sensors hold potential application in the real-time detection of O2â¢- in biological samples.
Assuntos
Técnicas Biossensoriais , Grafite/química , Ácido Fítico/química , Superóxidos/isolamento & purificação , Carbono/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Células HeLa , Humanos , Limite de Detecção , Óxidos/química , Superóxidos/químicaRESUMO
Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiol peroxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed.
Assuntos
Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Peróxido de Hidrogênio/isolamento & purificação , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/patologia , NADPH Oxidases/metabolismo , Neoplasias/patologia , Oxirredução , Superóxidos/isolamento & purificação , Superóxidos/metabolismoRESUMO
Quantification of superoxide anions (O2â¢-) is significant in the monitoring of many serious diseases and the design of enzyme-mimic catalysts plays the main role in the development of non-enzymatic O2â¢- sensors. Herein, we proposed a facile self-assembly process to synthesize manganese phosphate modified carbon networks using three kinds of widely-used carbon materials (MWCNTs, NGS and GO) as pillar connectors. Characterizations demonstrate that manganese phosphate is widely dispersed inside and on the surface of carbon networks without visible morphology. Meanwhile, all three kinds of synthesized catalysts were successfully immobilized on the screen-printed carbon electrodes to evaluate the electrochemical performance of fabricated sensors. The results indicate that sensors based on Mnx(PO4)y modified MWCNTs exhibit high sensitivity with an extremely low detection limit of 0.127µM (S/N = 3) and a wide liner range of 0-1.817mM (R2 = 0.998). We further employed the recommended sensors in the real-time monitoring of HepG2 cells released O2â¢- under the stimulating of Zymosan (20mg/mL). Noticeably, the proposed sensors exhibit not only sensitive response but also stable current steps upon different addition of Zymosan. The calculated concentrations of cell-released O2â¢- vary from 6.772 to 24.652pM cell-1 for the Zymosan amount used in this work. The established novel sensors display low background current and signal noises, thus holding unique advantages in the trace analysis of O2â¢- in biological samples and in vivo environment.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Superóxidos/isolamento & purificação , Catálise , Enzimas Imobilizadas/química , Células Hep G2 , Humanos , Limite de Detecção , Nanotubos de Carbono/química , Compostos Organometálicos/química , Superóxidos/químicaRESUMO
A novel non-enzymatic superoxide anion (O2â¢-) sensor was fabricated based on Ag nanoparticles (NPs)/L-cysteine functioned carbon nanotubes (Cys-MWCNTs) nanocomposites and used to measure the release of O2â¢- from living cells. In this strategy, AgNPs could be uniformly electrodeposited on the MWCNTs surface with average diameter of about 20nm as exhibited by scanning electronmicroscopy (SEM). Electrochemical study demonstrated that the AgNPs/Cys-MWCNTs modified glassy carbon electrode exhibited excellent catalytic activity toward the reduction of O2â¢- with a super wide linear range from 7.00×10-11 to 7.41×10-5M and a low detection limit (LOD) of 2.33×10-11M (S/N=3). Meanwhile, the mechanism for O2â¢- reduction was also proposed for the first time. Importantly, this novel non-enzymatic O2â¢- sensor can detect O2â¢- release from cancer cells under both the external stimulation and the normal condition, which has the great potential application in clinical diagnostics to assess oxidative stress of living cells.
Assuntos
Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Neoplasias/diagnóstico , Superóxidos/isolamento & purificação , Cisteína/química , Humanos , Peróxido de Hidrogênio/química , Nanocompostos/química , Nanotubos de Carbono/química , Neoplasias/patologia , Estresse Oxidativo/genética , Prata/química , Superóxidos/químicaRESUMO
Reactive oxygen species (ROS) play a critical role in cell signaling and disease pathogenesis. Despite their biological importance, assessment of ROS often involves measurement of indirect byproducts or measurement of ROS from excised tissue. Herein, we describe a microdialysis technique that utilizes the Amplex Ultrared assay to directly measure hydrogen peroxide (H2O2) and superoxide in tissue of living, anesthetized rats and mice. We demonstrate the application of this methodology in the penis, adipose tissue, skeletal muscle, kidney, and liver. We provide data demonstrating the impact of important methodological considerations such as membrane length, perfusion rate, and time-dependence upon probe insertion. In this report, we provide a complete list of equipment, troubleshooting tips, and suggestions for implementing this technique in a new system. The data herein demonstrate the feasibility of measuring both in vivo H2O2 and superoxide in the extracellular environment of various rodent tissues, providing a technique with potential application to a vast array of disease states which are subject to oxidative stress.
Assuntos
Peróxido de Hidrogênio/isolamento & purificação , Microdiálise/métodos , Espécies Reativas de Oxigênio/isolamento & purificação , Superóxidos/isolamento & purificação , Animais , Peróxido de Hidrogênio/química , Camundongos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/química , Roedores , Superóxidos/químicaRESUMO
Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions.
Assuntos
Técnicas Biossensoriais/métodos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/isolamento & purificação , Superóxidos/isolamento & purificação , Animais , Proteínas de Bactérias/química , Transporte Biológico , Humanos , Proteínas Luminescentes/química , Microscopia Confocal/métodos , Espécies Reativas de Oxigênio/química , Transdução de Sinais , Superóxidos/químicaRESUMO
Detection of superoxide produced by living cells has been an on-going challenge in biology for over forty years. Various methods have been proposed to address this issue, among which spin trapping with cyclic nitrones coupled to EPR spectroscopy, the gold standard for detection of radicals. This technique is based on the nucleophilic addition of superoxide to a diamagnetic cyclic nitrone, referred to as the spin trap, and the formation of a spin adduct, i.e. a persistent radical with a characteristic EPR spectrum. The first application of spin trapping to living cells dates back 1979. Since then, considerable improvements of the method have been achieved both in the structures of the spin traps, the EPR methodology, and the design of the experiments including appropriate controls. Here, we will concentrate on technical aspects of the spin trapping/EPR technique, delineating recent breakthroughs, inherent limitations, and potential artifacts.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres/isolamento & purificação , Detecção de Spin/métodos , Superóxidos/isolamento & purificação , Radicais Livres/química , Óxidos de Nitrogênio/química , Marcadores de Spin , Superóxidos/químicaRESUMO
Redox processes play a key role in cells of all.organisms. These processes imply directed flows of electrons via so-called redox pairs: substances that exist in both reduced and oxidized states simultaneously within the cell. Examples of redox pairs are NAD+/NADH, NADP+/NADPH, GSSG/2GSH. Until recently, studies of redox processes in the living cells were challenged by the lack of suitable methods. Genetically encoded fluorescent biosensors provide a new way to study biological processes including redox ones. Biosensors allow real-time detection of messengers, metabolites and enzymatic activities in living systems of different complexity from cultured cells to transgenic animals. In this review, we describe the main types of known redox biosensors with examples of their use.
Assuntos
Técnicas Biossensoriais , Peróxido de Hidrogênio/isolamento & purificação , Oxirredução , Superóxidos/isolamento & purificação , Animais , Dissulfeto de Glutationa/química , Proteínas de Fluorescência Verde/química , Peróxido de Hidrogênio/química , NAD/química , NADP/química , Espécies Reativas de Oxigênio/química , Superóxidos/químicaRESUMO
Monitoring of reactive oxygen species like superoxide anion (O2(â-)) turns to be of increasing significance considering their potential damages to organism. In the present work, we fabricated a novel O2(â-) electrochemical sensor through immobilizing superoxide dismutase (SOD) onto a Pt-Pd/MWCNTs hybrid modified electrode surface. The Pt-Pd/MWCNTs hybrid was synthesized via a facile one-step alcohol-reduction process, and well characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. The immobilization of SOD was accomplished using a simple drop-casting method, and the performance of the assembled enzyme-based sensor for O2(â-) detection was systematically investigated by several electrochemcial techniques. Thanks to the specific biocatalysis of SOD towards O2(â-) and the Pt-Pd/MWCNTs - promoted fast electron transfer at the fabricated interface, the developed biosensor exhibits a fast, selective and linear amperometric response upon O2(â-) in the concentration scope of 40-1550 µM (R(2)=0.9941), with a sensitivity of 0.601 mA cm(-2) mM(-1) and a detection limit of 0.71 µM (S/N=3). In addition, the favorable biocompatibility of this electrode interface endows the prepared biosensor with excellent long-term stability (a sensitivity loss of only 3% over a period of 30 days). It is promising that the proposed sensor will be utilized as an effective tool to quantitatively monitor the dynamic changes of O2(â-) in biological systems.
Assuntos
Técnicas Biossensoriais , Enzimas Imobilizadas/química , Superóxido Dismutase/química , Superóxidos/isolamento & purificação , Eletrodos , Humanos , Nanotubos de Carbono/química , Espectroscopia Fotoeletrônica , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/isolamento & purificação , Superóxidos/química , Difração de Raios XRESUMO
TAT-SOD is a recombinant protein of superoxide dismutase fused with TAT peptide. By pure accident, we discovered that topical application of TAT-SOD to acupoints could result in acupuncture-like action. This study aimed to validate the accidental discovery by investigating the effect on simple obesity of the topical application of TAT-SOD to acupoints in comparison with acupuncture. 90 subjects were divided into 3 groups for 12-week treatments. Regular hospital acupuncture treatment was given to Acupuncture Group 3 times a week. TAT-SOD Group were instructed first to locate acupoints and apply 0.1ml of 5000u SOD/ml TAT-SOD cream in an area of 1cm(2) to each of the same set of acupoints, which they then conducted at home three times daily. Placebo Group applied the vehicle cream the same manner as TAT-SOD Group. Both TAT-SOD and acupuncture treatments decreased adiposity with overall clinical effective rates of 60.0% and 76.7%, respectively. The placebo group showed no improvement. The results validate that the enzymatic removal of the intracellular superoxide at acupoints could generate acupuncture-like effects, and indicate a possibility of the new method as a simple substitute to acupuncture and an insight of superoxide modulation along meridians for acupuncture mechanism.
Assuntos
Pontos de Acupuntura , Produtos do Gene tat/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , Acupuntura , Adulto , Produtos do Gene tat/administração & dosagem , Humanos , Valores de Referência , Superóxido Dismutase/administração & dosagem , Superóxidos/isolamento & purificação , Adulto JovemRESUMO
The radical anion of dioxygen superoxide (O2.-) is a physiological free radical formed in various enzymatic processes. On the one hand superoxide is a precursor of reactive oxygen and nitrogen species (hydroxyl radicals, peroxy radicals, hydrogen peroxide, peroxynitrite, etc.), -the initiators of cellular damage; on the other hand it is a signaling molecule regulating numerous physiological processes including apoptosis, aging, and senescence. Therefore, the detection and measurement of superoxide in cells, tissues, and whole organisms is of a vital importance for in vitro and in vivo studies of many physiological and pathophysiological processes. At present different efficient methods were developed, which allow to identificate and measure superoxide in biological systems. In present review the credibility and efficiency of principal mostly applied methods of superoxide detection based on one-electron transfer and nucleophilic reactions are discussed, and spectrophotometrical, chemiluminescent, fluorescent, and ESR spin trapping methods are compared.
Assuntos
Medições Luminescentes/métodos , Oxigênio/metabolismo , Espectrofotometria/métodos , Detecção de Spin/métodos , Superóxidos/química , Superóxidos/isolamento & purificação , Acridinas , Citocromos c/metabolismo , Imidazóis , Luminol/análogos & derivados , Pirazinas , Superóxidos/metabolismoRESUMO
This work presents findings, which indicate important role of fructose, fructose 6-phosphate (F6P), and fructose 1,6-bisphosphate (FBP) in preservation of homeostasis in plants under low temperature. Cold combined with light is known to incite increased generation of superoxide in chloroplasts leading to photoinhibition, but also an increased level of soluble sugars. In the present study, oxidative stress in pea leaves provoked by cold/light regime was asserted by the observed decrease of the level of oxidized form of PSI pigment P700 (P700+). Alongside, the increased antioxidative status and the accumulation of fructose were observed. The antioxidative properties of fructose and its phosphorylated forms were evaluated to appraise their potential protective role in plants exposed to chilling stress. Fructose, and particularly F6P and FBP exhibited high capacities for scavenging superoxide and showed to be involved in antioxidative protection in pea leaves. These results combined with previously established links implicate that the increase in level of fructose sugars through various pathways intercalated into physiological mechanisms of homeostasis represents important non-enzymatic antioxidative defense in plants under cold-related stress.
Assuntos
Adaptação Fisiológica , Temperatura Baixa , Frutose/metabolismo , Estresse Oxidativo , Fenômenos Fisiológicos Vegetais , Plantas/metabolismo , Antioxidantes/metabolismo , Clorofila/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Homeostase , Pisum sativum/metabolismo , Pisum sativum/fisiologia , Superóxidos/isolamento & purificação , Superóxidos/metabolismoRESUMO
We have previously reported the oxidation of kojic acid catalyzed by manganese peroxidase (MnP) from Ceriporiopsis subvermispora. This reaction is strictly dependent on Mn(II), although it does not require the addition of hydrogen peroxide. We have extended these studies because this reaction can be considered as a model system for the in situ generation of hydrogen peroxide in natural environments. We show here that oxidation of kojic acid with horseradish peroxidase (HRP) plus hydrogen peroxide or with manganic acetate rendered a product with identical chromatographic and spectral properties as the one obtained in the reaction catalyzed by MnP. The initial lag observed in the latter reaction decreased significantly upon UV irradiation of the substrate. On the other hand, ascorbic acid increased the lag and did not affect the yield of the reaction. The superoxide anion trapping agents glutathione, nitroblue tetrazolium, and superoxide dismutase markedly affected the reaction. In contrast, addition of the hydroxyl radical scavengers mannitol and salicylic acid had no effect. Based on these results, a mechanism for the MnP-catalyzed reaction is proposed.
Assuntos
Basidiomycota/metabolismo , Peroxidases/metabolismo , Pironas/metabolismo , Ácido Ascórbico/metabolismo , Cromatografia Líquida de Alta Pressão , Radicais Livres/antagonistas & inibidores , Radicais Livres/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/metabolismo , Manganês/metabolismo , Oxirredução , Peroxidases/química , Pironas/química , Espectrofotometria Ultravioleta , Superóxidos/isolamento & purificação , Raios UltravioletaRESUMO
Some important immunoprotective effects of human breast milk have been attributed to the presence of macrophages. We investigated the generation of superoxide anion (O2-) by monocytes and human milk macrophages after stimulation with opsonized and unopsonized zymosan in the absence and presence of mannose as an inhibitor to investigate lectinophagocytic and opsonophagocytic properties. Peripheral blood monocytes generated more O2- than human milk macrophages (417,4 + 79,1 nmol O2-/mg protein vs. 216,1 +/-15,1 nmol O2-/mg protein, p<0,05) after stimulation with opsonized zymosan. When unopsonized zymosan was used as a serum-independent stimulus monocytes generated slightly less O2- in comparison to human milk macrophages (150,8 +/- 34,5 nmol/mg protein vs. 176,1 +/- 18 nmol O2-/mg protein, p<0,05). These findings demonstrate that the proportion of opsonin-independent phagocytosis in human milk macrophages is higher than in monocytes (82% vs. 36%). When mannose was used as an inhibitor a significantly higher reduction of O2- generation occurred in human milk macrophages compared to monocytes stimulated with opsonized zymosan, whereas no difference was found when unopsonized zymosan was used. These results indicate that human milk macrophages are stimulated to a greater extent by opsonin-independent mechanisms than blood borne monocytes. As the colostrum and the intestinal environment of the neonate offers only a little amount of opsonins like complement and immunoglobulin G, such a differentiation to lectinophagocytic properties could bear a great advantage for protective functions of human milk macrophages.
Assuntos
Lectinas Tipo C , Macrófagos/imunologia , Lectinas de Ligação a Manose , Leite Humano/imunologia , Monócitos/imunologia , Fagocitose , Superóxidos/metabolismo , Adjuvantes Imunológicos , Contagem de Células , Feminino , Humanos , Lectinas/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Manose/farmacologia , Receptor de Manose , Leite Humano/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteínas Opsonizantes/imunologia , Fagocitose/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Superóxidos/isolamento & purificação , Zimosan/administração & dosagemRESUMO
Garlic has been reported to provide protection against hypercholesterolemic atherosclerosis and ischemia-reperfusion-induced arrhythmias and infarction. Oxygen free radicals (OFRs) have been implicated as causative factors in these diseases and antioxidants have been shown to be effective against these conditions. The effectiveness of garlic in these disease states could be due to its ability to scavenge OFRs. However, the OFR-scavenging activity of garlic is not known. Also it is not known if its activity is affected by cooking. We therefore investigated, using high pressure liquid chromatography, the ability of garlic extract (heated or unheated) to scavenge exogenously generated hydroxyl radical (.OH). .OH was generated by photolysis of H2O2 (1.2-10 mumoles/ml) with ultraviolet (UV) light and was trapped with salicylic acid (500 nmoles/ml). H2O2 produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. Garlic extract (5-100 microliters/ml) produced an inhibition (30-100%) of 2,3-DHBA and 2,5-DHBA generated by photolysis of H2O2 (5.00 pmoles/ml) in a concentration-dependent manner. Its activity is reduced by 10% approximately when heated to 100 degrees C for 20, 40 or 60 min. The extent of reduction in activity was similar for the three heating periods. Garlic extract prevented the .OH-induced formation of malondialdehyde in the rabbit liver homogenate in a concentration-dependent manner. It alone did not affect the MDA levels in the absence of .OH. These results indicate that garlic extract is a powerful scavenger of .OH and that heating reduces its activity slightly.
Assuntos
Sequestradores de Radicais Livres , Alho , Gentisatos , Radical Hidroxila , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Extratos Vegetais/farmacologia , Plantas Medicinais , Animais , Cromatografia Líquida de Alta Pressão , Temperatura Alta , Hidroxibenzoatos , Malondialdeído/análise , Malondialdeído/metabolismo , Fotólise , Coelhos , Salicilatos , Ácido Salicílico , Sensibilidade e Especificidade , Superóxidos/isolamento & purificação , Superóxidos/metabolismo , Raios UltravioletaRESUMO
This study examined effects of blood-contacting materials on the monocyte reaction following the first contact of human blood with hollow fibre dialyser membranes under pyrogen-free conditions. Membrane materials were the unchanged regenerated cellulose, the synthetic polysulphone (PS), a positively charged diethylaminoethyl cellulose (DEAE-C), the negatively charged carboxymethyl cellulose (CMC) and acrylonitrile copolymer (AN). The experimental system involved perfusion with human fresh venous blood through different modules containing the materials in the form of hollow fibre membranes. Extracellular and intracellular aspects of blood reactions after the first contact with the materials were investigated in Ficoll-separated granulocytes and peripheral blood mononuclear cells. Investigations were done by release reactions of platelet activating factor (PAF), oxygen radical (O2-), leukotriene B4, prostaglandin E2 (PGE2) and cytokines (IL-1 beta, TNF-alpha, IL-6). The intracellular activation of peripheral blood mononuclear cells was done by mRNA transcription of IL-1 beta, TNF-alpha, IL-6, IL-8 and beta 2-microglobulin (beta 2-MG). From the set of parameters, release reactions were only measurable for PAF, PGE2 and O2- if a second stimulus (phorbol myristate acetate, lipopolysaccharide, zymosan and calcium ionophore) was used after blood-membrane interaction. Although the extent of the release reaction was weak, negatively charged membranes were, in general, more active. All dialysers exhibited the same increase in beta 2-MG mRNA transcription, suggesting that all blood-contacting membranes initiate the gene expression of beta 2-MG at the same level. TNF-alpha, IL-6, IL-1 beta and IL-8 mRNAs were demonstrated in the AN and CMC membranes rather than the other materials, which exhibit a lower transcription than the tubing set. As has been found, an enhanced generation of PGE2 for both CMC and AN membranes supports, therefore, the concept of an effect of the negative charges of the materials in the gene expression of cytokines. However, this initiation does not lead to the generation of cytokines, even after stimulation with pyrogens.
Assuntos
Materiais Biocompatíveis , Citocinas/farmacologia , Granulócitos/imunologia , Membranas Artificiais , Monócitos/imunologia , Diálise Renal , Animais , Humanos , Leucotrieno B4/metabolismo , Teste de Materiais , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , Coelhos , Superóxidos/isolamento & purificação , Superóxidos/metabolismo , Transcrição Gênica , Microglobulina beta-2/metabolismoRESUMO
Human neutrophils possess a superoxide (O2-)-forming NADPH oxidase which is activated by the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), complement C5a, platelet-activating factor and leukotriene B4. We studied the roles of cAMP and cGMP in the regulation of O2- formation using the cell-permeant analogues of cyclic nucleotides, N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) and N2,2'-O-dibutyryl guanosine 3':5'-cyclic monophosphate (Bt2cGMP). Bt2cAMP inhibited O2- formation induced by these chemoattractants to similar extents. Bt2cGMP as low as 10 mumol/l significantly inhibited O2- formation induced by fMet-Leu-Phe at a submaximally effective concentration (50 nmol/l), and Bt2cGMP was more effective in diminishing O2- formation than Bt2cAMP. In contrast, Bt2cGMP did not affect O2- formation induced by fMet-Leu-Phe at a maximally effective concentration (1 mumol/l). Bt2cGMP (0.1 and 1 mmol/l) enhanced O2- formation induced by 0.1 mumol/1 C5a by 23% and 49%, respectively, and Bt2cGMP antagonized inhibition of O2- formation caused by Bt2cAMP. Bt2cGMP inhibited platelet-activating factor-induced O2- formation to a lesser extent than Bt2cAMP and had no effect on that induced by leukotriene B4. Bt2cAMP and Bt2cGMP had no effect on O2- formation induced by NAF, gamma-hexachlorocyclohexane, phorbol myristate acetate, A 23187 and arachidonic acid. Our data suggest that: 1. Bt2cAMP generally inhibits chemoattractant-stimulated O2- formation. 2. Bt2cGMP inhibits fMet-Leu-Phe- and platelet-activating factor-stimulated O2- formation but potentiates C5a-induced O2- formation.(ABSTRACT TRUNCATED AT 250 WORDS)