Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques. (c) 2006 Elsevier B.V. All rights reserved.

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 mu M) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8 '-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH*) and superoxide (O-2(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H2O2 accumulation and Fe2+ -citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O-2(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H2O2. (C) 2007 Elsevier Ltd. All rights reserved.

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains 100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9). (c) 2006 Elsevier B.V.. All rights reserved.

Metabolismo mitocondrial, radicais livres e envelhecimento

O envelhecimento pode estar associado ao maior acúmulo de lesões celulares decorrentes das espécies reativas do oxigênio e do nitrogênio derivadas do metabolismo mitocondrial. Com a progressão da idade, há acúmulo de proteínas, lipídeos, carboidratos e DNA oxidados em relação a organismos jovens, de acordo com a teoria dos radicais livres. Entretanto, nem sempre os idosos ou animais envelhecidos apresentam maior estresse oxidativo que os jovens. Este artigo discute o paradoxo da teoria dos radicais livres de acordo com a teoria da biogênese da manutenção adequada do metabolismo mitocondrial. Diversos fatores podem contribuir para a redução do estresse oxidativo, como a hormese induzida pela prática regular de exercícios físicos, a restrição calórica, a ingestão de antioxidantes nutricionais e o aumento da produção de antioxidantes celulares que. em conjunto, estes promovem a expressão das sirtuínas e das proteínas do choque térmico, protegendo a integridade e funcionalidade mitocondriais, reduzindo o estresse oxidativo e nitrosativo, o que está associado à redução do envelhecimento e aumento da longevidade.

Fisioterapia respiratória em crianças com pneumonia: revisão sistemática

Introduction: Pneumonia is an inflammatory lung disease and it is the greatest cause of deaths in children younger than five years of age worldwide. Chest physiotherapy is widely used in the treatment of pneumonia because it can help to eliminate inflammatory exudates and tracheobronchial secretions, remove airway obstructions, reduce airway resistance, enhance gas exchange and reduce the work of breathing. Thus, chest physiotherapy may contribute to patient recovery as an adjuvant treatment even though its indication remains controversial. Objectives: To assess the effectiveness of chest physiotherapy in relation to time until clinical resolution in children (from birth up to 18 years old) of either gender with any type of pneumonia. Methods: We searched CENTRAL 2013, Issue 4; MEDLINE (1946 to May week 4, 2013); EMBASE (1974 to May 2013); CINAHL (1981 to May 2013); LILACS (1982 to May 2013); Web of Science (1950 to May 2013); and PEDro (1950 to May 2013). We consulted the ClinicalTrials.gov and the WHO ICTRP registers to identify planned, ongoing and unpublished trials. We consulted the reference lists of relevant articles found by the electronic searches for additional studies. We included randomised controlled trials (RCTs) that compared chest physiotherapy of any type with no chest physiotherapy in children with pneumonia. Two review authors independently selected the studies to be included in the review, assessed trial quality and extracted data. Results: Three RCTs involving 255 inpatient children are included in the review. They addressed conventional chest physiotherapy, positive expiratory pressure and continuous positive airway pressure. The following outcomes were measured: duration of hospital stay, time to clinical resolution (observing the following parameters: fever, chest indrawing, nasal flaring, tachypnoea and peripheral oxygen saturation levels), change in adventitious sounds, change in chest X-ray and duration of cough in days. Two of the included studies found a significant improvement in respiratory rate and oxygen saturation whereas the other included study failed to show that standardised respiratory physiotherapy and positive expiratory pressure decrease the time to clinical resolution and the duration of hospital stay. No adverse effects related to the interventions were xvi described. Due to the different characteristics of the trials, such as the duration of treatment, levels of severity, types of pneumonia and the techniques used in children with pneumonia, as well as differences in their statistical presentation, we were not able to pool data. Two included studies had an overall low risk of bias whereas one included study had an overall unclear risk of bias. Conclusion: Our review does not provide conclusive evidence to justify the use of chest physiotherapy in children with pneumonia due to a lack of data. The number of included studies is small and they differed in their statistical presentation

Associação entre polimorfismos em enzimas de reparo de DNA e ocorrência de meningite bacteriana.

Despite advances in vaccine development and therapy, bacterial meningitis (BM) remains a major cause of death and long-term neurological disabilities. As part of the host inflammatory response to the invading pathogen, factors such as reactive oxygen species are generated, which may damage DNA and trigger the overactivation of DNA repair mechanisms. It is conceivable that the individual susceptibility and outcome of BM may be in part determined by non synonymous polymorphisms that may alter the function of crucial BER DNA repair enzymes as PARP-1, OGG-1 and APE-1. These enzymes, in addition to their important DNA repair function, also perform role of inflammatory regulators. In this work was investigated the non synonymous SNPs APE-1 Asn148Glu, OGG-1 Ser326Cys,PARP-1 Val762Ala, PARP-1 Pro882Leu and PARP-1 Cys908Tyr in patients with bacterial meningitis (BM), chronic meningitis (CM), aseptic meningitis (AM) and not infected (controls). As results we found increased frequency of variant alleles of PARP-1 Val762Ala (P = 0.005) and APE-1 Asn148Glu (P=0.018) in BM patients, APE-1 Asn148Glu in AM patients (P = 0.012) and decrease in the frequency of the variant allele OGG-1 Ser326Cys in patients with CM (P = 0.013), regarding the allelic frequencies in the controls. A major incidence of individuals heterozygous and/ or polymorphic homozygous in BM for PARP-1 Val762Ala (P= 0.0399, OD 4.2, 95% IC 1.213 -14.545) and PARP-1 Val762Ala/ APE-1 Asn148Glu (P = 0.0238, OD 11.111, 95% IC 1.274 - 96.914) was observed related to what was expected in a not infected population. It was also observed a major incidence of combined SNPs in the BM patients compared with the control group (P=0.0281), giving evidences that SNPs can cause some susceptibility to the disease. This combined effect of SNPs seems to regulate the principal cytokines and other factors related to BM inflammatory response and point the importance of DNA repair not only to repair activity when DNA is damaged, but to others essential functions to human organism balance.

2-Bromo-1,4-naphthoquinone: a potentially improved substitute of menadione in Apatone therapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Interconverting flavanone glucosides and other phenolic compounds in Lippia salviaefolia Cham. ethanol extracts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

4-Fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito in vitro da terapia fotodinâmica sobre bactérias orais em suspensão e na formação de biofilme : ensaio com azul de metileno e toluidina ativados por luz halógena

Photodynamic therapy (PDT) has been proposed as an alternative method for the treatment of biofilm-dependent oral diseases like dental caries. This therapy consists of simultaneous action of a visible light (L) and a photosensitizer (FS) in the presence of oxygen, which leads to production of different reactive oxygen species that can interact with the bacterial cell components, and promote cell death. This study aims to evaluate the antimicrobial action of PDT on oral bacteria in suspension, as well as the formation of mono and multi-species biofilms, in vitro, from a standard strain of Streptococcus mutans (ATCC 25175) and saliva samples, respectively. The dye methylene blue (MB) and toluidine blue (TB) were used at a concentration of 100 mg/ L and activated by halogen light (600 to 750 nm) from a modified hand held photopolymerizer (Ultralux ®, Dabi Atlante, Ribeirão Preto , São Paulo, Brazil.). Planktonic cultures were prepared and submitted to different experimental conditions: 1. PDT using TB 2. PDT using MB, 3. L+ FS- , 4. TB + L - ; 5. MB+ L-; 6. L- FS- (negative control) and 7. administration of 0.12% chlorhexidine digluconate (positive control) (Periogard ®, Colgate-Palmolive Company, New York, USA). The immediate and mediated action of PDT on bacterial suspensions, as well as its effect on biofilm formation were observed from the number of colony-forming units per milliliter (CFU/mL) and measures optical density (OD). The data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney test for the significance level of 5%. According to the results, the PDT showed no antibacterial action on suspensions of S. mutans, regardless of the dye used. PDT with MB activated by halogen light was able to reduce 86.6% CFU/mL multi-species planktonic cultures, however, this reduction was not significant (p > 0.05). PDT showed antibacterial effect, mediate on multi-species planktonic cultures with TB (p < 0.001) and MB (p < 0.001), activated by halogen light. PDT was able to prevent the formation of multispecies biofilm, through the activation of TB by halogen light (p = 0.01). We conclude that activation of the dye toluidine blue and methylene blue, by halogen light (PDT) showed antimicrobial activity, compared to multi-species planktonic cultures prepared from saliva samples

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Genotoxicity and Fetal Abnormality in Streptozotocin-Induced Diabetic Rats Exposed to Cigarette Smoke Prior to and during Pregnancy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of leukocytospermia on the outcomes of assisted reproductive technology

Background: It is not well established whether the increased number of leukocytes in the seminal fluid impairs the outcomes of assisted reproductive technology (ART). This investigation analysed the outcomes of the intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) cycles in couples in which the male partner exhibited leukocytospermia.Methods: A total of 100 cycles in 100 couples were included in this study. For the ICSI or IMSI procedures, the patients were divided into two groups according to the presence or absence of leukocytospermia and then matched by (female) age:- ICSI: Group I (n = 25): Leukocytospermia - semen samples with a leukocyte count of greater than or equal to 1 x 10(6)/mL; and Group II (n = 25): Non-leukocytospermia - semen samples with a leukocyte count < 1 x 10(6)/mL.- IMSI: Group I (n = 25): Leukocytospermia; and Group II (n = 25): Non-leukocytospermia.The endpoints included the rates of fertilisation, implantation, clinical pregnancy, miscarriage, ongoing pregnancy and live birth. Student's t-tests, Mann-Whitney tests and Chi-square tests were performed, and P < 0.05 was considered significant.Results: The data from the ICSI groups showed that leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 57.9+/-30.2%, Group II: 61.9+/-27.7%; P = 0.74), implantation (Group I: 12.3%; Group II: 13.5%; P = 0.93), clinical pregnancy (Group I: 24%; Group II: 24%; P = 1.0), miscarriage ( Group I: 0, Group II: 0), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%; Group II: 24%; P = 1.0). Similarly, the data from the IMSI groups also showed that the leukocytospermia did not have a negative influence on the rates of fertilisation (Group I: 67.6+/-24.6%, Group II: 59.5+/-28.1%; P = 0.36), implantation (Group I: 17.5%; Group II: 16.7%; P = 0.90), clinical pregnancy (Group I: 28%; Group II: 24%; P = 1.0), miscarriage (Group I: 14.3%; Group II: 0; P = 0.33), ongoing pregnancy (Group I: 24%; Group II: 24%; P = 1.0), or live births (Group I: 24%, 6/25; Group II: 24%, 6/25; P = 1.0).Conclusions: The results indicate that the leukocytospermia may not have a negative effect on the outcomes of ICSI or IMSI cycles. Nevertheless, it seems that it is necessary to more precisely determine the effects, if any, of seminal leukocytes on fertilisation and implantation processes. Such efforts will help to establish a more reliable leukocyte threshold, which could eventually demonstrate whether there is a negative influence on the ART procedures.

Comprovação do efeito antioxidante de plantas medicinais utilizadas no tratamento do Diabetes mellitus em animais: artigo de atualização

Diabetes mellitus (DM) é uma síndrome de etiologia múltipla caracterizada por hiperglicemia crônica. Esta hiperglicemia induz o aumento na produção de espécies reativas de oxigênio (ERO) e diminuição das defesas antioxidantes. Devido às complicações causadas pelo diabete, muitos indivíduos optam por terapias alternativas à base de plantas medicinais para amenizar seus efeitos. Sendo assim, nesta revisão de literatura, foram analisados e descritos diversos trabalhos experimentais com a utilização de animais diabéticos para comprovar os efeitos antioxidantes de algumas dessas plantas e verificar se os títulos e resumos disponibilizados nos artigos são compatíveis aos objetivos de nossa busca.

Espécies reativas do oxigênio e as doenças respiratórias em grandes animais

As espécies reativas do oxigênio (ERO) são moléculas instáveis e extremamente reativas capazes de transformar outras moléculas com as quais colidem. As ERO são geradas em grande quantidade durante o estresse oxidativo, condição em que são afetadas moléculas como proteínas, carboidratos, lipídeos e ácido nucleicos. Neste trabalho, são discutidos os principais conceitos sobre os radicais livres e as ERO: principais tipos, sua formação e a forma como atuam sobre as estruturas celulares, provocando lesão tecidual significativa. Os principais sistemas de defesa antioxidantes e a influência do aumento na produção dessas ERO no trato respiratório de grandes animais também são discutidos, dando ênfase ao envolvimento das ERO em doenças como a pneumonia em ruminantes e na obstrução recorrente das vias aéreas e a hemorragia pulmonar induzida por exercício em equinos.