Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N-2-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of Sao Sebastiao (Sao Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n = 76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n = 7), V. alginolyticus (n = 8), V. campbellii (n = 3), and V parahaemolyticus (n = 1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N, might explain why they are so abundant in the mucus of different coral species. (C) 2008 Published by Elsevier GmbH.

Comparison of two methods for collecting free-living ticks in the Amazonian forest

In the present study, we compared 2 methods for collecting ixodid ticks on the verges of animal trails in a primary Amazon forest area in northern Brazil. (i) Dragging: This method was based on passing a 1-m(2) white flannel over the vegetation and checking the flannel for the presence of caught ticks every 5-10 m. (ii) Visual search: This method consisted of looking for guesting ticks on the tips of leaves of the vegetation bordering animal trails in the forest. A total of 103 adult ticks belonging to 4 Amblyomma species were collected by the visual search method on 5 collecting dates, while only 44 adult ticks belonging to 3 Amblyomma species were collected by dragging on 5 other collecting dates. These values were statistically different (Mann-Whitney Test, P = 0.0472). On the other hand, dragging was more efficient for subadult ticks, since no larva or nymph was collected by visual search, whereas 18 nymphs and 7 larvae were collected by dragging. The visual search method proved to be suitable for collecting adult ticks in the Amazon forest: however, field studies should include a second method, such as dragging in order to maximize the collection of subadult ticks. Indeed, these 2 methods can be performed by a single investigator at the same time, while he/she walks on an animal trail in the forest. (C) 2010 Elsevier GmbH. All rights reserved.

Phylogenetic Validation of the Genera Angomonas and Strigomonas of Trypanosomatids Harboring Bacterial Endosymbionts with the Description of New Species of Trypanosomatids and of Proteobacterial Symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia cuiicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history. (C) 2011 Elsevier GmbH. All rights reserved.

Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein

The clearance of apoptotic cells by phagocytes is a fundamental process during tissue remodeling and resolution of inflammation. In turn, the phagocytosis of apoptotic cells generates signals that suppress pro-inflammatory activation of macrophages. These events occur during the resolution phase of inflammation and therefore the malfunctioning of this process may lead to inflammation-related tissue damage. Here, we demonstrate that the calcium-binding protein S100A9, normally abundant in the cytoplasm of neutrophils and also released by apoptotic neutrophils, is involved in the suppression of macrophages after the uptake of apoptotic neutrophils. Both, spontaneous and induced production of inflammatory species (nitric oxide, hydrogen peroxide and TNF-alpha) as well as the phagocytic activity were inhibited when macrophages were in presence of apoptotic neutrophils, conditioned medium from neutrophil cultures or a peptide corresponding to the C-terminal region of S100A9 protein. On the other hand, macrophages kept in the conditioned medium of neutrophils that was previously depleted of S100A9 were shown to resume the activated status. Finally, we demonstrate that the calcium-binding property of S100A9 might play a role in the suppression process, since the stimulation of intracellular calcium release with ionomycin significantly reversed the effects of the uptake of apoptotic neutrophils in macrophages. In conclusion, we propose that S100A9 is a novel component of the regulatory mechanisms of inflammation, acting side-by-side with other suppressor factors generated upon ingestion of apoptotic cells. (C) 2009 Elsevier GmbH. All rights reserved.

Wave optics analysis by phase-shifting real-time holographic interferometry

The purpose of this work is to study the potentialities in the phase-shifting real-time holographic interferometry using photorefractive crystals as the recording medium for wave-optics analysis in optical elements and non-linear optical materials. This technique was used for obtaining quantitative measurements from the phase distributions of the wave front of lens and lens systems along the propagation direction with in situ visualization, monitoring and analysis in real time. (C) 2008 Elsevier GmbH. All rights reserved.

Lacustrine sediments provide geochemical evidence of environmental change during the last millennium in southeastern Brazil

A 172 cm-long sediment core was collected from a small pristine lake situated within a centripetal drainage basin in a tropical karst environment (Ribeira River valley, southeastern Brazil) in order to investigate the paleoenvironmental record provided by the lacustrine geochemistry. Sediments derived from erosion of the surrounding cambisoils contain quartz, kaolinite, mica, chlorite and goethite. Accelerator mass spectroscopy (AMS) (14)C dating provided the geochronological framework. Three major sedimentary units were identified based on the structure and color of the sediments: Unit III from 170 to 140 cm (1030 +/- 60-730 +/- 60 yr BP), Unit II from 140 to 90 cm (730 +/- 60-360 +/- 60 yr BP) and Unit I from 90 to 0 cm (360 +/- 60-0 yr BP). Results of major and trace element concentrations were analysed through multivariate statistical techniques. Factor analysis provided three factors accounting for 72.4% of the total variance. F1 and F2 have high positive loadings from K, Ba, Cs, Rb, Sr, Sc, Th, light rare earth element (LREE), Fe, Cr, Ti, Zr, Hf and Ta, and high negative loadings from Mg, Co, Cu, Zn, Br and loss on ignition (LOI). F3, with positive loadings from V and non-metals As and Sb, accounts for a low percentage (9.7%) of the total variance, being therefore of little interpretative use. The profile distribution of F1 scores reveals negative values in Units I and III, and positive values in Unit II, meaning that K, Ba, Cs, Rb, Sr, Sc, Th, LREE, Fe, Cr, Ti, Zr, Hf and Ta are relatively more concentrated in Unit II, and Mg, Co, Cu, Zn and Br are relatively more abundant in Units I and III. The observed fluctuations in the geochemical composition of the sediments are consistent with slight variations of the erosion intensity in the catchment area as a possible response to variations of climatic conditions during the last millennium. (c) 2009 Elsevier GmbH. All rights reserved.

Effects of inhaled Linalool in anxiety, social interaction and aggressive behavior in mice

Aromatherapy uses essential oils (EOs) for several medical purposes, including relaxation. The association between the use of aromas and a decrease in anxiety could be a valuable instrument in managing anxiety in an ever increasing anxiogenic daily life style. Linalool is a monoterpene commonly found as the major volatile component of EOs in several aromatic plant species. Adding to previously reported sedative effects of inhaled linalool, the aim of this study was to investigate the effects of inhaled linalool on anxiety, aggressiveness and social interaction in mice. Additionally, we investigated the effects of inhaled linalool on the acquisition phase of a step-down memory task in mice. Inhaled linalool showed anxiolytic properties in the light/dark test, increased social interaction and decreased aggressive behavior; impaired memory was only seen the higher dose of linalool. These results strengthen the suggestion that inhaling linalool rich essential oils can be useful as a mean to attain relaxation and counteract anxiety. (C) 2009 Elsevier GmbH. All rights reserved.

In vitro basal cytotoxicity assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite

Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite. the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 mu M) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 mu M, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies. (C) 2010 Elsevier GmbH. All rights reserved.

Inhaled linalool-induced sedation in mice

Linalool is a monoterpene often found as a major component of essential oils obtained from aromatic plant species., many of which are used in traditional medical systems as hypno-sedatives. Psychopharmacological evaluations of linalool (i.p. and i.c.v.) revealed marked sedative and anticonvulsant central effects in various mouse models. Considering this profile and alleged effects of inhaled lavender essential oil, the purpose of this study was to examine the sedative effects of inhaled linalool in mice. Mice were placed in an inhalation chamber during 60 min, in an atmosphere saturated with 1% or 3% linalool. Immediately after inhalation, animals were evaluated regarding locomotion, barbiturate-induced sleeping time, body temperature: and motor coordination (rota-rod test). The 1% and 3% linalool increased (p < 0.01) pentobarbital sleeping time and reduced (p<0.01) body temperature. The 3% linalool decreased (p<0.01) locomotion. Motor coordination was not affected. Hence, linalool inhaled for I h seems to induce sedation without significant impairment in motor abilities, a side effect shared by most psycholeptic drugs. (C) 2008 Elsevier GmbH. All rights reserved.

ACE inhibition by astilbin isolated from Erythroxylum gonocladum (Mart.) OE Schulz

Erythroxylum species have several traditional uses in different countries, including the treatment of hypertension. The ethanol extract from E. gonocladum aerial parts, a species endemic to the Brazilian cerrado, elicited a concentration-dependent inhibition of angiotensin converting enzyme (ACE) (pIC(50)=4.53 +/- 0.05). Extract fractionation led to the isolation of two compounds, whose structures were assigned by spectrometric data as astilbin and beta-sitosterol, along with a mixture of palmitic, stearic and linolenic acids. This is the first report on the occurrence of these compounds on E. gonocladum. Astilbin promoted significant ACE inhibition in vitro (pIC(50)=5.86 +/- 0.33) and its activity did not differ from captopril, when both compounds were assayed at 10 mu M concentration. (C) 2009 Elsevier GmbH. All rights reserved.

Microbial diversity associated with algae, ascidians and sponges from the north coast of Sao Paulo state, Brazil

Little is known about the microbial diversity associated with marine macroorganisms, despite the vital role microorganisms may play in marine ecosystems. The aim of the present study was to investigate the diversity of bacteria and fungi isolated from eight marine invertebrate and one algae samples. Data derived from ARDRA and sequencing analyses allowed the identification of marine-derived microorganisms isolated from those samples. Microbial strains identified up to the genus level revealed 144 distinct ribotypes out of 256 fungal strains and 158 distinct ribotypes out of 181 bacterial strains. Filamentous fungi were distributed among 24 different genera belonging to Ascomycota, Zygomycota and Basidiomycota, some of which had never been reported in the literature as marine invertebrate-inhabiting fungi (Pestalotiopsis, Xylaria, Botrysphaeria and Cunnninghamella). Bacterial isolates were affiliated to 41 different genera, being Bacillus, Ruegeria, Micrococcus, Pseudovibrio and Staphylococcus the most abundant ones. Results revealed an unexpected high microbial diversity associated to the macroorganisms which have been collected and suggested the selection of certain microbial taxonomic groups according to the host. The combined data gathered from this investigation contribute to broaden the knowledge of microbial diversity associated to marine macroorganisms, including as a promising source for the discovery of new natural products. (C) 2009 Elsevier GmbH. All rights reserved.

Stomach fullness modulates prey size choice in the frillfin goby, Bathygobius soporator

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

Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants

Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, degrades starch, this degradation being supposed to occur in the plant material which leafcutters forage to the nests, generating most of the glucose which the ants utilize for food. In the present investigation, we show that laboratory cultures of L. gongylophorus produce extracellular alpha-amylase and maltase which degrade starch to glucose, reinforcing that the ants can obtain glucose from starch through the symbiotic fungus. Glucose was found to repress a-amylase and, more severely, maltase activity, thus repressing starch degradation by L. gongylophorus, so that we hypothesize that: (1) glucose down-regulation of starch degradation also occurs in the Atta sexdens fungus garden; (2) glucose consumption from the fungus garden by A. sexdens stimutates degradation of starch from plant material by L. gongylophorus, which may represent a mechanism by which Leafcutters can control enzyme production by the symbiotic fungus. Since glucose is found in the fungus garden inside the nests, down-regulation of starch degradation by glucose is supposed to occur in the nest and play a part in the control of fungal enzyme production by leafcutters. (c) 2005 Elsevier GmbH. All rights reserved.

New features of Cuban endemic Pinguicula filifolia (Lentibulariaceae) and considerations on its habitat and ecology

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.