Gallic acid as the major antioxidant in pequi (Caryocar brasiliense Camb. ) fruit peel / Ácido gálico como principal antioxidante da casca do fruto do pequi (Caryocar brasiliense Camb. )

ABSTRACTIn this study, ethanol-water extracts of pequi fruit peel were fractionated in order to identify and quantify the major antioxidant present in it. The fractions were subjected to liquid-liquid phase extraction and silica-gel column chromatography, and antioxidant activity was monitored using the 2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay. The purity of the fractions was evaluated using thin-layer chromatography and high-performance liquid chromatography (HPLC). The substance with antioxidant property was identified through the analysis in a liquid chromatography-mass spectroscopy fragmentation and was quantified using HPLC. After the Silica-gel fractionation, it was identified a fraction with high antioxidant activity and purity, which contained gallic acid as the main compound. The gallic acid was found at the amount of 26.54 ± 1.13 mg/g of the dry mass of the pequi fruit peel. Because the quantifications were performed using crude ethanol-water extract, it was suspected that gallic acid was present in a free form. Thus, pequi fruit peel may serve as an attractive alternative of feedstock for natural antioxidant production. Moreover, the results obtained in this study emphasize the value of the pequi plant, and suggests improved opportunities for families that use this fruit`s products.

RESUMOExtratos hidroetanólicos da casca do fruto do pequi foram fracionados a fim de identificar e quantificar o principal antioxidante presente. Frações do extrato foram submetidas ao particionamento líquido-líquido e fracionamento em coluna de sílica gel. As atividades antioxidantes das frações foram monitoradas usando ensaio de redução do radical 2,2-difenil-1-picrilhidrazila e a pureza das frações foi avaliada em cromatografia de camada delgada e cromatografia líquida de alta eficiência (CLAE). A substância com propriedades antioxidantes foi identificada através da análise em sistema de cromatografia líquida associada à espectrometria de massas e foi quantificada em HPLC. Após o fracionamento identificou-se uma fração com alta atividade antioxidante e pureza, contendo ácido gálico como o composto principal. Ácido gálico foi encontrado em concentrações de 26,54 ± 1,13 mg/g de massa seca. Devido às quantificações terem sido realizadas no extrato hidroetanólico bruto, acredita-se que o ácido gálico esteja presente na forma livre. Assim, a casca do fruto pequi pode servir como interessante alternativa de matéria prima para a produção desse antioxidante natural. Além disso, esse resultado enfatiza o valor da planta do pequi e sugere oportunidades para as famílias que utilizam produtos de pequi.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age.

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98 th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin.

AIMS: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. METHODS AND RESULTS: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1.3x10(-8) mol l(-1), failed in the detection of some of these isolates. CONCLUSION: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. SIGNIFICANCE AND IMPACT OF THE STUDY: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

To evaluate the sensitivity and specificity of polyclonal and monoclonalantibodies (Mabs) against intimin in the detection of enteropathogenic andenterohaemorrhagic Escherichia coli isolates using immunoblotting.Polyclonal and Mabs against the intimin-conservedregion were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of1Æ3 · 10)8 mol l)1, failed in the detection of some of these isolates. All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Biofilm on the apical region of roots in primary teeth with vital and necrotic pulps with or without radiographically evident apical pathosis.

AIM: To evaluate, by scanning electron microscopy (SEM), the presence of biofilms on the external surfaces of the apical third of roots of human primary teeth with vital or necrotic pulps with and without radiographically evident periradicular pathosis. METHODOLOGY: Eighteen teeth were selected: group I - normal pulp (n = 5), group II - pulp necrosis without radiographic evidence of periapical pathosis (n = 7) and group III - pulp necrosis with well-defined radiographic periapical pathosis (n = 6). After extraction, the teeth were washed with saline and immersed in 0.03 g mL(-1) trypsin solution for 20 min. The teeth were then washed in sodium cacodilate buffer and stored in receptacles containing modified Karnovsky solution. The teeth were sectioned, dehydrated in an ethanol series, critical-point dried with CO(2), sputter coated with gold and the external root surface in the apical third examined by SEM. RESULTS: In the teeth of groups I and II, the apical root surfaces were covered by collagen fibres, with no evidence of bacteria (100%). In the teeth of group III, the root apices had no collagen fibres but revealed resorptive areas containing microorganisms (cocci, bacilli, filaments and spirochetes) in all cases (100%). CONCLUSION: Microorganisms organized as biofilms on the external root surface (extraradicular infection) were detected in primary teeth with pulp necrosis and radiographically visible periapical pathosis.

Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition.

AIMS: To determine the suitability of eight different commercial broth media for Shiga toxin (Stx) production. METHODS AND RESULTS: Shiga toxin-producing Escherichia coli (STEC) strains producing Stx1 or Stx2 were grown at 37 degrees C (250 rev min(-1)) for 24 h in brain heart infusion broth, E. coli broth, Evans medium, Luria-Bertani broth, Penassay broth, buffered-peptone water, syncase broth and trypticase soy broth. Toxin production was measured by enzyme-linked immunosorbent assay (ELISA) in polymyxin-treated cell pellets and/or supernatants of cultures, ELISA optical densities reached 1 when isolates were grown for 2-4 h in E. coli broth in the presence of antibiotic. Besides, a collection of STEC-expressing Stx strains was evaluated and the Stx production was assayed in the supernatants and in polymyxin-treated pellets of bacterial growth after 4 h of cultivation in E. coli broth in the presence of antibiotic. CONCLUSIONS: The most suitable medium for Stx production was E. coli broth when the bacterial isolates were grown for 4 h in the presence of ciprofloxacin and the Stx production is detected in the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first comprehensive comparison of different broth media with regard to Stx production to establish optimal culture conditions for STEC detection in routine diagnostic laboratories.

Morphometric analysis of experimentally induced periapical lesions: radiographic vs histopathological findings.

OBJECTIVES: To evaluate the accuracy and reliability of conventional (Kodak Ektaspeed Plus film) and digitized radiographic images to detect the presence as well to estimate the size, as measured by an image analysis programme, of periapical radiolucencies induced in dog teeth in comparison with the histomorphometric data obtained from the same lesions by conventional and fluorescence microscopy. METHOD: After the removal of pulp, the root canals of five premolars from the same animal were left exposed for 7 days after which they were sealed for 60 days. At day 53, three more premolars were opened and left exposed to the oral cavity for 7 days. Intact premolars were used as control. Conventional radiographs were taken at day 0, day 7, day 30, day 45 and day 60. Morphometry in digitized radiographic images and histological sections were compared at day 7 and day 60 after setting the experimental series. RESULTS: Radiographically, periapical lesions were only detected 30 days after coronal sealing. A progressively increasing radiolucent lesion area was observed at day 45 and day 60. Histopathologically, 7 days after pulp removal dense inflammatory infiltrate and root resorption in the periapical region was observed. At day 7 and day 60, the lesion sizes were similar when evaluated by both conventional and fluorescence microscopy. Lesion size was about 20% larger in digitized radiographs in comparison with histological measurements. CONCLUSIONS: Although image digitization could not improve the detection of the early stages of periapical lesions, it provides a valuable quantitative assessment of extensive periapical lesions. In addition, fluorescence light microscopy enhances the visualization of the apical and periapical structures and seems to be a highly useful tool for histological evaluation, valuable for both qualitative and quantitative studies of periapical disease.

Mitochondrial damage as an early event of monensin-induced cell injury in cultured fibroblasts L929.

The present study was designed to identify, submicroscopically, the primary organelle or target structure for monensin in cultured murine fibroblasts L929. In addition, the effect of the drug on cell size and surface membranes of the cells were analysed; cellular proliferation, collagen secretion, and necrosis and apoptosis were re-evaluated. At the lowest concentration of monensin the foremost ultrastructural alteration occurred in the mitochondria, characterized by increased matrix density with disorganized and less distinct crystae. Incubation with monensin at higher concentrations resulted in severe mitochondrial damage and marked dilatation of the Golgi apparatus and rough endoplasmic reticulum cisternae. Fibroblasts exposed to higher concentrations of monensin were enlarged with decreased number of filopodia and hollows in the surface membrane. Moreover, monensin inhibited the cell proliferation, increased immunohistochemical positiveness for collagen type I in a dose-dependent manner, and, at high concentrations, caused cell necrosis whereas apoptosis was not induced. Taken together, these results show that monensin induces early mitochondrial damage, possibly causing an energy deficit that led to inhibition of fibroblasts proliferation and accumulation of collagen causing dilatation of Golgi apparatus and rough endoplasmic reticulum. Moreover, the mitochondrial damage would also explain the monensin-induced necrosis.

Analysis of susceptibility profile of Pseudomonas spp. and prevalence of bacterial samples from the surfaces of dental consulting-rooms

The aim of this research was to evaluate the susceptibility profile of Pseudomonas spp. and the prevalence of bacterial samples isolated from horizontal surfaces surrounding wash-basins used by dentists in several adjoined consulting-rooms, at points next to and at a distance from the basin, before and after surgical procedures. Our results showed a high percentage of Gram-positive cocci and Gram-negative bacilli; 34.66% were Staphylococcus spp. and 30.12% were non-fermentative Gram-negative bacilli among which Pseudomonas spp. (40.90%) was the commonest genus. Analysis of the susceptibility profile of Pseudomonas spp. isolates by determining the minimal inhibitory concentration (MIC) of 14 antibiotics showed a great variation among the strains and high rates of resistance to cefazolin, ceftazidime and aztreonan. Of the 14 antibiotics tested, 59.03% were found to be active against all the environmental isolates. Strains were resistant to aztreonan (62.82%), while susceptibility to third generation cephalosporins was variable