Agreement between direct, oscillometric and Doppler ultrasound blood pressures using three different cuff positions in anesthetized dogs

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

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier B.V. All rights reserved.

Non-steroidal anti-inflammatory drugs may modulate the protease activity of Candida albicans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Atividade antagônica in vitro de isolados de Trichoderma spp. ao fungo Phytophthora citrophthora

Gummosis is among the main fungal diseases of the citrus. It is caused by Phytophthora sp. and usually shows up in the lap of the plant, provoking rottenness and gum exudation, and expands causing the plant death for constrictions in the cambium or phloem which interrupts the descending flow of sap. The objective of this work was to evaluate the antagonistic in vitro activity of Trichoderma spp. to the fungi Phytophthora citrophthora. Phytophthora citrophthora was exposed to five environments of antagonism (without antagonist and with four strains of Trichoderma viride, T. virens, T. harzianu and T stromaticum), The in vitro essay was accomplished through the method of paired cultures. A completely randomized desing was used with five treatments and three replications, and each plot was represented by three petri dishes. The isolates of Trichoderma demonstrated significant effect in the inhibition of the mycelial growth of the fungi Phytophthora citrophthora, and the fungi Trichoderma stromaticum presented larger antagonism to the fungi P. citrophthora while the T harzianum presented antagonism smaller.

Anatomical changes induced by selection for plant architecture in maize (Zea mays L.)

The original brachytic population 'Dent Single Cross Composite' (DSCC-br2br2) and a selection-derived sub-population with modified plant architecture (DSCC-br2br2-Lg3Lg3, selected for erect leaves), were evaluated for the following characteristics number of vascular bundles of greater and smaller size, total vascular tissue area (phloem and xylem), sustaining tissue area (vascular tissue plus sclerenchyma), phloem and sclerenchyma areas in apical, medial and basal portions from midclub and in apical and basal sheath regions (from second leaf above and first below ear insertion). These variables had different values for the five different sections studied in each leaf and these differences did not have the same pattern in the two DSCC populations (brachytic and with modified architecture). Selection for architectural modification caused some indirect foliar anatomical modifications. With the exception of the phloem and the vascular tissue areas in apical leaf and sheath base regions, the modified plant architecture population showed smaller values of sustaining tissue area, sclerenchyma area, vascular tissue area and number of smaller vascular bundles than the original one. In the ligule region the modified maize leaves had smaller vascular and sustaining tissue areas, reducing transportation area, which could reduce gram yield.

Distribution of Xylella fastidiosa in citrus rootstocks and transmission of citrus variegated chlorosis between sweet orange plants through natural root grafts

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

Drought tolerance of two field-grown clones of Coffea canephora

We compared tolerance to soil drought of two field-grown clones of Coffea canephora (clone 46, drought-sensitive; and clone 120, drought-tolerant). Under irrigation, there were no marked differences between the clones in water relation parameters, gas exchange and total leaf area. Under rainfed conditions, clone 46 showed osmotic adjustment and increased tissue rigidity. These adjustments, however, were incapable of preventing substantial decreases in xylem pressure potential. By contrast, clone 120 did not exhibit osmotic adjustment, but was able to increase tissue elasticity and to maintain xylem pressure potentials to a greater extent than clone 46 (despite having twice the total leaf area of this clone). Stomatal conductance was lowered by drought in clone 120 but not in clone 46. Carbon assimilation per unit leaf area in both clones remained unaffected under stress. Long-term water use efficiency (WUE), as estimated through carbon isotope discrimination, was consistently greater in clone 120 than in clone 46. Because of these traits, clone 120 was better able to postpone dehydration and to maintain whole-tree photosynthesis. It is proposed that these features should decisively contribute to buffer its productivity in drought-prone areas. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Little cicada of sugarcane Mahanarva posticata (Homoptera: Cercopidae). A Brazilian agricultural pest. Morpho-histological study of salivary glands

Little cicadas are homopteran insect pests of sugarcane plantations. As these insects suck out the sap from the leaf parenchyma, they inoculate a toxic saliva that damages the plant vessels, thus promoting the loss of glucose by the affected plant. The morphological and histological analyses of the salivary glands of the little cicada Mahanarva posticata, revealed that these glands are formed by 2 portions: one portion comprises a group of acini and has been denominated as the principal gland; the second portion is filamentous in nature and has been denominated as the accessory gland; it is formed by very long and fine filaments. The acinous portion of the gland can be subdivided into 2 lobes: an anterior lobe formed by 3 lobules (I, II, III), and a posterior lobe formed by lobule IV and the excretory duct. Histologically, the salivary glands showed that the filaments are empty sutructures composed by several internal channels with secretion granules being observed in the cytoplasm of the cells of the secretory filaments. Lobules I and II of the principal gland are characterized by being highly basophilic and for accumulating a large amount of secretion in both the cytoplasm of the cells and inside secretion vesicles. Histochemically, we verified that the secretion produced by these glands is lipidic and protein in nature, with the production of polysaccharides being very low. The differences in stain and appearance of the different regions of the salivary gland lead us to believe that the final glandular product is lipoproteic in nature.

Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings

Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Effect of storage of cuttings on the rooting of different cultivars of cut chrysanthemums (Dendranthema grandiflora Tzvelev) for two different seasons

Chrysanthemums are commercially propagated through cuttings. Pre-rooting storage of cuttings in the dark is a common practice among growers and companies that work and trade with chrysanthemum cuttings. Therefore, the maximum storage period for cuttings and differences in tolerance among cultivars has been investigated. Adventitious roots of cuttings can originate in almost any tissue, including the epidermis, stem cortex and pericycle, ray parenchyma, immature xylem and phloem cells, and pith. The aims of this work were to determine the effect of time of cold storage of cuttings (0, 1, 2, 3, 4, 5 and 6 weeks) on the rooting of four cut chrysanthemum cultivars (Super White, Sheena, Dark Orange Reagan and Town Talk) for two seasons of the year (summer and winter), and also to determine the origin of root formation in chrysanthemum cuttings. The study was carried out as a randomized complete block design with 5 replications for each storage treatment. Each plot was comprised of three cuttings that were examined 14 days after the cutting procedure. During the winter, the roots were studied by scanning electron microscopy (SEM) at the Electron Microscope Laboratory. The following conclusions were made: in winter, cold storage affected the rooting of cuttings, mainly after two weeks of storage for all cultivars. The rooting percentage was lower in the winter and the cuttings could be preserved for a shorter period. The source and growth of roots in chrysanthemum cuttings was found to be endogenous. After three days, callus formed in the pericycle and the first emergence of adventitious roots occurred by the fourth day of rooting. During the summer, cold storage could be up to 4 weeks without any problems.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Oral toxicity of chemical substances found in Dimorphandra mollis (Caesalpiniaceae) against honeybees (Apis mellifera) (Hymenoptera: Apidae)

Dimorphandra mollis is a characteristic plant from Brazilian saP vanna-like vegetation. The pollen of this species could be toxic to bees and the objective of the present investigation was to study the toxicity of methanolic extracts obtained from the flowers, peduncles and stem bark of D. mollis to Apis mellifera workers. For the study, the extracts were incorporated into the diet of the bees for later evaluation of mortality rates. The substances isolated: neoisoastilbin, catechin, astilbin and tannins were tested on adult workers and only catechin did not cause toxic effects. The data obtained in the toxicity bioassays were analyzed statistically by Log Rank test and all methanolic extracts showed significant (p<0.0001) toxic effects. Astilbin is also the major component of pollen grains, and is probably responsible for honeybee mortality during blooming periods.

Efetividade de um programa de exercícios no condicionamento físico, perfil metabólico e pressão arterial de pacientes hipertensos

Background and objective: It has been shown that aerobic exercise is useful to reduce arterial pressure, however, the effectiveness of an exercise program is still controversial and not very well analyzed among populations with low-income. The objective of the present study was to set up an individualized physical fitness program - Projeto Hipertensão - focused on hypertensive people, patients from a Health Basic Unit (HBU) and, after that, to investigate the effects of this program on physical fitness, metabolic profile and pressure levels. Methods: Sixteen hypertensive women (56 ± 3yrs) under regular pharmacological treatment underwent 4 months of a supervised aerobic and stretching exercise program (3 sessions/wk, 90 min/session, 60% of V̇O 2 max). Several physical and metabolic variables were compared before and after 4 months of training. Results: Training significantly reduced systolic arterial pressure (SAP, -6%), improved cardio-respiratory fitness (+42% of V̇O2max), flexibility (+11%) and plasma glucose content (-4%). BMI and % fat did not change. Besides modifying metabolic profile, it was found that training presented significant correlations between individual initial values of cholesterol total level (CT), high density lipoprotein (HDL-C) and low density lipoprotein (LDL-C) and its responses after exercise. Conclusions: The study shows that exercise programs can be personalized for hypertensive patients from a HBU and confirms the effectiveness of exercise on AP, physical fitness, flexibility and lipid profile on hypertensive patients. The expressive reduction of AP in hypertensive subjects suggests that this exercise intervention should be emphasized on other health centers which assist low-income population.

Morfo-anatomia do eixo vegetativo aéreo de Achyrocline alata (Kunth) DC. (Asteraceae)

Achyrocline alata occurs in a dense and low vegetation in Mato Grosso do Sul State, Brazil and it has been studied under physical-chemical and yield aspects, being scarce anatomical data. The present work has as objective the study of the aerial vegetative axis. Stem is cylindrical and hairy, having five wings, which are arisen from their leaves. The blade shows the presence of uniseriate epidermis, which is covered by the cuticle of variable thickness; and non-glandular and glandular trichomes. The non-glandular trichomes are uniseriate and multicellular and have their apex cell in whip form, while the glandular trichomes are multicellular, uniseriate or biseriate. The mesophyll is dorsiventral with uni-stratified palisade parenchyma in most of the cases and lacunal parenchyma formed by three to four layers of irregular cells. Only one collateral vascular bundle occurs in the midvein. Stem in transversal section is covered by epidermis with trichomes similar to leaves; the cortex is constituted by a discontinuous area of angular collenchyma, which is followed by chlorophyll parenchyma. Vascular cylinder that is delimited by pericycle shows vascular bundles of collateral type. In the secondary structure, the periderm is originated from epidermal and subepidermal tissues. In vascular region, the fascicular cambium differs into secondary xylem and phloem, while interfascicular cambium produces sclerenchymatous tissue.