Control of Rhizopus stolonifer in strawberries by the combination of essential oil with carboxymethylcellulose.

Strawberry has a limiting postharvest shelf life, especially because of soft rot. The antifungal activity of the essential oils (EOs) of Eucalyptus staigeriana, Lippia sidoides and Pimenta pseudocaryophyllus was tested in vitro against plant pathogen Rhizopus stolonifer. The chemical composition of the EO with the highest activity and its effects on pathogen morphology were verified. The in vivo antifungal activity of this EO associated with carboxymethylcellulose (CMC) coating, in preventive and curative applications, was also evaluated. L. sidoides EO presented the highest in vitro antifungal activity. The analysis of the chemical composition of this EO showed a prevalence of the compound thymol and the scanning and transmission electron microscopy showed that L. sidoides EO was able to cause damage to the cell wall and the intracellular components of the pathogen. Strawberries treated with L. sidoides EO associated with CMC presented a reduction in disease severity, especially when treated in a curative way.

Alimentação e prática de atividades físicas de crianças: hábitoscotidianos e culturais / Food and activities of children’s physical practice: daily habits and culture

Objetivo: conhecer os hábitos cotidianos de alimentação e prática de atividades físicas de crianças entre sete e dez anos de idade, de uma unidade básica de educação de um município do Sul de Santa Catarina. Método: pesquisa qualitativa descritiva e exploratória, desenvolvida com 24 crianças em uma unidade de ensino, vinculada a Estratégia de Saúde da família. Os dados foram coletados no período de outubro de 2012, por meio de entrevista semiestruturada e analisados a luz do referencial teórico de Madeleine Leininger. Resultados: o cotidiano das crianças e de suas famílias nem sempre auxiliam nos hábitos saudáveis, padrões culturais podem influenciar neste processo, consequentemente refletindo no ambiente escolar. Conclusão: parcerias entre a Estratégia de Saúde da Família (ESF), escola e família são importantes para traçar estratégias que fomentam as práticas de educação em saúde desta comunidade.

Objective: to learn about the everyday habits of food and physical activity of children from sevento ten years of age, from a basic unit of education at a city in the south of Santa Catarina. Methods:qualitative descriptive and exploratory study, developed with 24 children in a unit of education,linked to the Family Health Strategy. The data was collected in October 2012, through semistructuredinterviews, and analyzed through the theoretical framework of Madeleine Leininger.Results: the daily life of children and their families do not always assist in healthy habits; culturalpatterns may influence this process, consequently reflecting in the school environment. Conclusion:partnerships among the Family Health Strategy (FHS), school, and family are important to devisestrategies that foster the practices of health education in this community.

Physicochemical and sensory characteristics of fat-free goat milk yogurt with added stabilizers and skim milk powder fortification.

Goat milk yogurt has a less consistent coagulum compared with cow milk yogurt; furthermore, the presence of goat milk in foodstuffs imparts a characteristic flavor that can restrict its acceptance by consumers. This study aimed to assess and compare the physicochemical and sensory characteristics of fat-free goat milk yogurts with added stabilizers or bovine skim milk powder to improve the final product. Four treatment additions were evaluated: (1) a mixture of 0.1% (wt/vol) carrageenan and 0.1% (wt/vol) pectin (treatment CR); (2) 0.5% (wt/vol) pectin (treatment PE); (3) 4.65% (wt/vol) bovine skim milk powder (treatment BM); and (4) control (no stabilizer; treatment CT). The physicochemical parameters were investigated at on d 1 and 5 of storage. The BM treatment presented higher pH and titratable acidity values, resulting in a buffering capacity effect. The total crude protein (CP) and solids-not-fat (SNF) contents were also higher in BM compared with the other evaluated treatments because of the addition of bovine skim milk powder. We detected a reduction in pH values for all treatments. Lower SNF contents were present in the CR and CT treatments, which might be related to a syneresis process during storage; moreover, an increase in total CP was observed for all treatments due to the proteolytic action of the starter culture. Sensory attributes, including appearance (color, consistency, and presence of lumps), texture (consistency, viscosity, and presence of lumps), flavor (bitter, sweet, and characteristic of commercial plain nonfat yogurt), and overall impression were evaluated by quantitative descriptive analysis. The addition of 0.5% (wt/vol) of pectin (PE treatment) strengthened the curd; however, the visual and oral presence of lumps and a higher bitterness score were noted by trained panelists, which resulted in the lowest overall impression score for the PE treatment. In several sensory attributes, the CR treatment was considered similar to the control; the mixture of 0.1% (wt/vol) carrageenan and 0.1% (wt/vol) pectin was not as effective as expected. Goat milk yogurt containing added bovine skim milk powder (BM) had improved consistency, viscosity, and flavor due to its higher SNF and total CP contents, which are particularly important for the desirable texture of plain nonfat yogurt. In addition, the BM yogurt was considered to have characteristics most similar to that of available commercial brands and achieved the best score for overall impression.

Genetic variability in the ITS and IGS regions of the ribosomal DNA of Acremonium cavaraeanum exhibiting antimicrobial activity.

Endophytic microorganisms represent promising alternatives for obtaining new drugs of biotechnological importance. In this study, the endophytic species Acremonium cavaraeanum (A1a) isolated from Cocos nucifera was cultivated for the production of secondary metabolites, and its extracts and fractions were evaluated by the dilution method (MIC). The EtOAc extracts and MeOH fractions were tested against Gram-positive and -negative bacteria, and had an MIC of 125 µg/mL when evaluated in the EtOAc extract (EBI). The EtOAc extract (EBII) had an MIC of 62.25 µg/mL for Staphylococcus aureus and an MIC between 125 and 250 µg/mL for Gram-negative bacteria. The methanolic fractions showed activity with MIC between 125 and 250 µg/mL for all bacteria tested. The IGS region of the rDNA repeat unit of genomic DNA was analyzed by PCR/RFLPs, including endonucleases PstI, BamHII, HinfI, and EcoRI. The physical maps showed different restriction sites for the 6 Acremonium sp isolates, and revealed 5 RFLP patterns. The results showed that isolates of the same Acremonium species exhibited variation in this specific region. The sequences of ITS1-5.8S-ITS2 regions were aligned by Clustal W using the neighbor joining method, which grouped the isolates into 5 distinct clusters. This study aimed to evaluate the genetic diversity of A. cavaraeanum crops exhibiting antibacterial activity. The results of this study indicate that different fungal genetic isolates have biotechnological potential for the production of active bio-compounds against several human pathogens.

[Intracranial pressure monitoring as a complementary tests for diagnosing brain death. Preliminary observation through the report of 2 cases]. / Monitorização da pressão intracraniana como exame complementar no diagnóstico de morte encefálica. Observação preliminar através do registro de dois casos.

The clinical evolution of two patients with continuous intracranial pressure (ICP) monitoring, admitted to the Intensive Care Unit of Neurology, University Hospital of Botucatu, and followed until irreversible cardiac failure (ICF) was studied retrospectively. The evolution of ICP showed that it reached a maximum 5 to 12 hours before a decrease in wave amplitude occurred (this was observed approximately 47 to 60 hours before ICF). The tracing became linear approximately 30 hours before ICF in both cases. The clinical diagnosis of brain death (BD) was obtained 3 to 28 hours after the tracing had become linear. The authors suggest that, in absence of sedation, the diagnosis of BD may be made early with the use of ICP monitoring even before the clinical diagnosis, and emphasize the need for more observations in a larger number of patients.

Numerous cell targets for gastrin in the guinea pig stomach revealed by gastrin/CCKB receptor localization.

The localization of the gastrin/CCKB receptor (GR) has recently become a subject of debate, especially since the publication of evidence for its presence in an unsuspected location, namely in the lamina propria, the submucosal layer of the stomach lining. Knowledge of the receptor localization is important because of the critical role of gastrin secretion and its trophic effects on the gastric epithelium. The present study, which utilizes immunohistochemistry and electron microscopy as primary tools, provides unequivocal data concerning the localization of GR in the guinea pig stomach. GR is expressed in parietal cells, on chief cells, and in previously unreported endocrine cells of the stomach. It is not found in the lamina propria. The predominant localization of the receptor in the endocrine cells is on the membranes of cytoplasmic electron-dense secretory granules. The positioning of these cells in the gastric glands suggests that they may be involved in the uptake of gastrin from the circulation. The distribution of GR implies that it may be involved in the regulation of various processes and may mediate various effects of gastrin in the stomach.

In vitro activity of the antimicrobial peptide magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis.

Magainins are peptide antibiotics with broad antibacterial and antiparasitic activities, originally extracted from the skin of Xenopus laevis. We investigated the effects of magainin 1 against Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis. Viability of purified protozoa was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to magainin 1, Bonamia viability was reduced in a dose-dependent manner. Within the peptide concentration of 500 micrograms/ml, the parasite viability was reduced by 94%. Electron microscopy showed membrane damage and release of cytoplasmic organelles in the injured Bonamia. The study of magainin 1 activity against Ostrea edulis hemocytes did not show any morphological change in the host cells, and the peptide did not impair the capabilities of hemocytes to produce chemiluminescence when stimulated to phagocytize zymosan particles. The possibility to genetically transform molluscs to generate disease-resistant organisms is currently under investigation. Antimicrobial peptides such as magainins may provide effective gene sequences to be manipulated.

Bcl-2 expressed using a retroviral vector is localized primarily in the nuclear membrane and the endoplasmic reticulum of chicken embryo fibroblasts.

A complementary DNA for human bcl-2 was cloned into the replication competent avian retrovirus vector RCASBP, and the resulting virus was used to express human Bcl-2 protein at high levels in chicken embryo fibroblasts. The expression of Bcl-2 did not transform or significantly alter the longevity of the chicken embryo fibroblasts in the presence of normal amounts of serum. However, the expression of Bcl-2 blocked c-Myc-induced apoptosis in these cells. Fractionation of the infected chicken embryo fibroblasts indicated that the protein was distributed equally between nuclear and high density cytoplasmic membranes. Immunofluorescence analysis by confocal microscopy and immunoelectron microscopy showed that the Bcl-2 protein was primarily associated with the nuclear membrane and with the endoplasmic reticulum. Reduced amounts of the protein were associated with other membranes in the cytoplasm. These data show that, in this system, the Bcl-2 protein associates with the nuclear membrane and intracytoplasmic membranes but is not preferentially associated with mitochondria.

Alterations in the intermediate layer of goldfish meninges during adaptation to darkness.

The morphological changes in the intermediate endomeningeal layer of the goldfish brain during light and dark adaptation were studied by freeze-fracture electron microscopy. During the different stages of adaptation no significant changes were found in the density of intramembrane particles and nuclear pores in these cells. The density of plasmalemmal vesicles in the meningocyte surface increased in the groups maintained in the dark for 48 and 72 h (maximum) and then decreased in the group maintained for 96 h in the dark to a basal level. There were also morphological changes in the junctional complexes. At the upper cell membranes (in contact with the outer layer) of meningocytes in the group maintained in the dark for 48 h, we found an increase in the surface occupied by gap junctions. In addition, gap junctions were absent in the lateral membranes of meningocytes from animals maintained in the dark for 72 h. The morphology of gap junctions in the group maintained in the dark for 96 h was similar to that of the control group. These results suggest that the cells of the teleost intermediate endomeningeal layer undergo important changes in activity during adaptative experiments.

The Met proto-oncogene mesenchymal to epithelial cell conversion.

Coexpression of the human Met receptor and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), in NIH 3T3 fibroblasts causes the cells to become tumorigenic in nude mice. The resultant tumors display lumen-like morphology, contain carcinoma-like focal areas with intercellular junctions resembling desmosomes, and coexpress epithelial (cytokeratin) and mesenchymal (vimentin) cytoskeletal markers. The tumor cells also display enhanced expression of desmosomal and tight-junction proteins. The apparent mesenchymal to epithelial conversion of the tumor cells mimics the conversion that occurs during embryonic kidney development, suggesting that Met-HGF/SF signaling plays a role in this process as well as in tumors that express both epithelial and mesenchymal markers.

Freeze-fracture immunocytochemical study of the expression of native and recombinant GABAA receptors.

To assess the density and distribution of native and recombinant GABAA receptors we used label-fracture and fracture-flip technologies combined with immunocytochemistry using monoclonal and polyclonal Abs directed against the extracellular domain of the GABAA receptor protein located in the freeze-fracture replicas. In cortical neurons there is a high density of GABAA receptors on both soma and dendrites with some areas were the density of receptors is higher, but there are no well defined clusters. In cerebellar granule cells most of the receptors are distributed in round clusters both in neurites and soma. In astroglial cells the receptor density is lower than in neurons and only occasionally they appear in clusters. In cells transfected with cDNAs encoding for various molecular forms of GABAA receptor subunits, the receptor density is moderate when cDNAs for alpha, beta and gamma subunits are cotransfected; however, on cells cotransfected with cDNAs for beta and gamma subunits the receptor density is significantly lower. Recombinant receptors appear randomly distributed and occasionally they aggregate in small groups.

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.

Câncer de pulmão e retardo no diagnóstico: análise de 300 casos / Lung cancer and the delay in the diagnosis: analysis of 300 cases

This is a prospective study involving 300 persons with lung cancer admitted to the "Arnaldo Vieira de Carvalho" Cancer Institute (ICAVC). The intention of the survey was to detect delay in diagnosis after the initial symptoms. THe authors tried to identify causes of this delay and its implications. Patients were asked about the day that the symptoms started, medical care and specialists sought, number of physicians seen and their diagnosis, also examinations carried out and referrals. Results showed that 78 of cases were seen firstly by general practitioners and 69.6 looked for medical assistance at least 30 days after the clinical beginning of the disease. Chest X-rays could identify only 9 cases (3) without symptoms. The most common clinical diagnoses were: pneumonia (20), neoplasia (19), bronchitis/emphysema (9.3) and tuberculosis (8). The number of first appointments seen by the Public Health Services and Contracted Private Hospital Network was 64.1 and the second appointment was 70. Only 24 (8) of the patients were referred to ICAVC just after their first appointment and 64.4 after the third. The time lost between the first appointment and the diagnosis was longer than 90 days in 55.7 of cases. These people needed to see 3 to 4 doctors (as an average) to obtain a positive diagnosis. The diagnostic techniques used more frequently were bronchoscopy (59.7) and fine needle lung biopsy (18.4) and the delay was 20 and 10 days on average, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

[Lung cancer and the delay in the diagnosis: analysis of 300 cases]. / Câncer de pulmão e retardo no diagnóstico: análise de 300 casos.

This is a prospective study involving 300 persons with lung cancer admitted to the "Arnaldo Vieira de Carvalho" Cancer Institute (ICAVC). The intention of the survey was to detect delay in diagnosis after the initial symptoms. THe authors tried to identify causes of this delay and its implications. Patients were asked about the day that the symptoms started, medical care and specialists sought, number of physicians seen and their diagnosis, also examinations carried out and referrals. Results showed that 78% of cases were seen firstly by general practitioners and 69.6% looked for medical assistance at least 30 days after the clinical beginning of the disease. Chest X-rays could identify only 9 cases (3%) without symptoms. The most common clinical diagnoses were: pneumonia (20%), neoplasia (19%), bronchitis/emphysema (9.3%) and tuberculosis (8%). The number of first appointments seen by the Public Health Services and Contracted Private Hospital Network was 64.1% and the second appointment was 70%. Only 24 (8%) of the patients were referred to ICAVC just after their first appointment and 64.4% after the third. The time lost between the first appointment and the diagnosis was longer than 90 days in 55.7% of cases. These people needed to see 3 to 4 doctors (as an average) to obtain a positive diagnosis. The diagnostic techniques used more frequently were bronchoscopy (59.7%) and fine needle lung biopsy (18.4%) and the delay was 20 and 10 days on average, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramacrophagic Mycobacterium avium bacilli are coated by a multiple lamellar structure: freeze fracture analysis of infected mouse liver.

We used freeze fracture electron microscopy to study the fine structure of Mycobacterium avium inside phagosomes of murine macrophages. M. avium-susceptible C57BL/6 mice were infected with M. avium by intraperitoneal inoculation of 10(8) viable bacilli. We studied the microanatomy of the mycobacteria in 3-month infections of mice, a situation in which bacillary multiplication is extensive. In these samples, freeze fracture revealed that intraphagosomal bacilli were surrounded by a multilamellar coat that was apposed to the cell wall. In thin sections, in contrast, the area corresponding to the coat showed no substructure and was electron transparent (the so-called electron-transparent zone that has been previously reported by others). The multiple lamellae resembled an onionlike assembly that was inserted in between the mycobacterial wall outer surface and the phagosomal membrane. Each lamella of the M. avium coat was made up of parallel straight fibrils with a width of 5 nm. A variable number of lamellae, sometimes up to 10 or more elements, coated individual bacilli. The multilamellar coat was absent around both extracellular M. avium and intramacrophagic M. avium after short-term (45-min) inoculation of mice. The supramolecular organization of the M. avium lamellar coat as viewed here by freeze fracture is similar to that of purified mycoside C (P. Draper, J. Gen. Microbiol. 83:431-433, 1974; K.-S. Kim, M.R.J. Salton, and L. Barksdale, J. Bacteriol. 125:739-743, 1976), a mycobacterial component currently known as glycopeptidolipid (W.W. Barrow and P.J. Brennan, J. Bacteriol. 150:381-384, 1982). We conclude that M. avium bacilli growing in macrophages are surrounded by multilamellar capsulelike structures that contain glycopeptidolipid molecules.

Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and label fracture immunocytochemistry survey.

Giardia lamblia undergoes surface antigenic variation. The ultrastructural location of antigens on four different variants was studied by label fracture and immunocytochemistry with four monoclonal antibodies (MAbs), each of which recognized the predominant variant in a particular clone. Each Giardia clone and its reacting MAb showed similar findings. The entire surface of the organism was covered by a surface coat which contained the variant surface protein. The surface coat was densely and uniformly labeled. Unreacting MAbs failed to label the surface. Label-fractured Giardia trophozoites exposed to reactive MAb revealed a planar distribution of reactivity with no discernible relationship to visualized intramembranous particles. Unexpectedly, some Giardia trophozoites lacked a surface coat and consequently failed to react with the appropriate MAb. The biological relevance of coatless Giardia trophozoites is unknown. These findings localize the variant antigens to the surface coat of the parasite and identify a minority of the population which lacks a surface coat.

Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C.

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 degrees C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.

Bimodal redistribution of surface transmembrane glycoproteins during Ca2+-dependent secretion (acrosome reaction) in boar spermatozoa.

We used the acrosome reaction of boar sperm cells to study the dynamics of surface transmembrane glycoproteins (TMG) during a secretory process. The acrosome reaction is the Ca2+-dependent fusion of a large cytoplasmic vesicle (the acrosome) with the overlying segment of the plasma membrane (acrosomal cap) that leads to the release of the acrosomal enzymes. After triggering the acrosome reaction in vitro (2 mM-CaCl2 in the presence of 10 microM-A23187), we used freeze-fracture electron microscopy to follow the topographical rearrangement of a population of acrosomal-cap large intramembrane particles that correspond to transmembrane proteins that bind wheat germ agglutinin. We found that these TMG move in the direction of either one of two opposite poles, proximal and distal, of the acrosomal cap. This bimodal movement of the TMG reorganizes the acrosomal cap into three extensive domains. The first two, on the apical rim and on the equator, are membrane domains to which the TMG are directed and where they accumulate. The third, a large in-between area of protein clearing, corresponds to the region from which TMG were preferentially located before displacement induced by the Ca2+ effect. The topography of these new membrane domains of the acrosomal cap becomes coincident with that of the structural domains of the subjacent acrosomal membrane. Mirroring of the acrosomal membrane by the plasma membrane is followed by fusion between the two membranes, formation of an exquisite labyrinth of hybrid-membrane tubules, followed by fission and release of the acrosomal contents through intertubular fenestrae.

Cell surface nanoanatomy of Leishmania major as revealed by fracture-flip. A surface meshwork of 44 nm fusiform filaments identifies infective developmental stage promastigotes.

Fracture-flip (Anderson-Forsman and Pinto da Silva, J. Cell Sci. 90, 531-541; 1988) was used to reveal the nanoanatomy of the surface of Leishmania major promastigotes. Over the cell surface of infective metacyclic promastigotes we identify a meshwork of 44 nm long, fusiform filaments. These filaments are not seen in noninfective stages of the parasite. Replica-staining immunocytochemistry with monoclonal antibody against infective metacyclic lipophosphoglycan shows a uniform distribution of protein A-colloidal gold complexes over the cell surface. Thin sections show that acquisition of the high molecular weight lipophosphoglycan is reflected in a thicker glycocalyx. Conventional freeze-fracture shows that in infective metacyclic promastigotes there is a reversal of the partition of intramembrane particles--an additional morphological marker for the infective developmental stage. We hypothesize that the fusiform filaments represent metacyclic developmental lipophosphoglycan.