Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans.

Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1-0.4 per thousand) and Bland-Altman analyses revealed no significant bias (0.2 per thousand). For further validation of this two-stage analyses strategy, all the specimens (n=124) collected during a major sport event were processed.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC-QTOFMS using a charged surface hybrid column.

INTRODUCTION: The analysis of glucosinolates (GS) is traditionally performed by reverse-phase liquid chromatography coupled to ultraviolet detection after a time-consuming desulphation step, which is required for increased retention. Simpler and more efficient alternative methods that can shorten both sample preparation and analysis are much needed. OBJECTIVE: To evaluate the feasibility of using ultrahigh-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) for the rapid profiling of intact GS. METHODOLOGY: A simple and short extraction of GS from Arabidopsis thaliana leaves was developed. Four sub-2 µm reverse-phase columns were tested for the rapid separation of these polar compounds using formic acid as the chromatographic additive. High-resolution QTOFMS was used to detect and identify GS. RESULTS: A novel charged surface hybrid (CSH) column was found to provide excellent retention and separation of GS within a total running time of 11 min. Twenty-one GS could be identified based on their accurate mass as well as isotopic and fragmentation patterns. The method was applied to determine the changes in GS content that occur after herbivory in Arabidopsis. In addition, we evaluated its applicability to the profiling of other Brassicaceae species. CONCLUSION: The method developed can profile the full range of GS, including the most polar ones, in a shorter time than previous methods, and is highly compatible with mass spectrometric detection.

In vitro heat exposure induces a redistribution of nuclear matrix proteins in human K562 erythroleukemia cells.

By using both conventional and confocal laser scanning microscopy with three monoclonal antibodies recognizing nuclear matrix proteins we have investigated by means of indirect fluorescence whether an incubation of isolated nuclei at the physiological temperature of 37 degrees C induces a redistribution of nuclear components in human K562 erythroleukemia cells. Upon incubation of isolated nuclei for 45 min at 37 degrees C, we have found that two of the antibodies, directed against proteins of the inner matrix network (M(r) 125 and 160 kDa), gave a fluorescent pattern different from that observed in permeabilized cells. By contrast, the fluorescent pattern did not change if nuclei were kept at 0 degrees C. The difference was more marked in case of the 160-kDa polypeptide. The fluorescent pattern detected by the third antibody, which recognizes the 180-kDa nucleolar isoform of DNA topoisomerase II, was unaffected by heat exposure of isolated nuclei. When isolated nuclear matrices prepared from heat-stabilized nuclei were stained by means of the same three antibodies, it was possible to see that the distribution of the 160-kDa matrix protein no longer corresponded to that observable in permeabilized cells, whereas the fluorescent pattern given by the antibody to the 125-kDa polypeptide resembled that detectable in permeabilized cells. The 180-kDa isoform of topoisomerase II was still present in the matrix nucleolar remnants. We conclude that a 37 degrees C incubation of isolated nuclei induces a redistribution of some nuclear matrix antigens and cannot prevent the rearrangement in the spatial organization of one of these antigens that takes place during matrix isolation in human erythroleukemia cells. The practical relevance of these findings is discussed.

Association of irisin with fat mass, resting energy expenditure, and daily activity in conditions of extreme body mass index.

FNDC5/irisin has been recently postulated as beneficial in the treatment of obesity and diabetes because it is induced in muscle by exercise, increasing energy expenditure. However, recent reports have shown that WAT also secretes irisin and that circulating irisin is elevated in obese subjects. The aim of this study was to evaluate irisin levels in conditions of extreme BMI and its correlation with basal metabolism and daily activity. The study involved 145 female patients, including 96 with extreme BMIs (30 anorexic (AN) and 66 obese (OB)) and 49 healthy normal weight (NW). The plasma irisin levels were significantly elevated in the OB patients compared with the AN and NW patients. Irisin also correlated positively with body weight, BMI, and fat mass. The OB patients exhibited the highest REE and higher daily physical activity compared with the AN patients but lower activity compared with the NW patients. The irisin levels were inversely correlated with daily physical activity and directly correlated with REE. Fat mass contributed to most of the variability of the irisin plasma levels independently of the other studied parameters. Conclusion. Irisin levels are influenced by energy expenditure independently of daily physical activity but fat mass is the main contributing factor.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Modelling changes in stable isotope compositions of minerals during net transfer reactions in a contact aureole: Wollastonite growth at the northern Hunter Mountain Batholith (Death Valley National Park, USA)

One of the world's largest wollastonite deposits was formed at the contact of the northern Hunter Mountain Batholith (California, USA) in Paleozoic sediments. Wollastonite occurs as zones of variable thickness surrounding layers or nodules of quartzite in limestones. A minimum formation temperature of 650 degrees C is estimated from isolated periclase-bearing lenses in that area. Contact metamorphism of siliceous carbonates has produced mineral assemblages that are consistent with heterogeneous, and partly limited infiltration of water-rich fluids, compatible with O-18/O-16 and C-13/C-12 isotopic patterns recorded in carbonates. Oxygen isotope compositions of wollastonites in the study area may also not require infiltration of large quantities of externally-derived fluids that were out of equilibrium with the rocks. 8180 values of wollastonite are high (14.8 parts per thousand to 25.0 parts per thousand; median: 19.7 parts per thousand) and close to those of the host limestone (19.7 parts per thousand to 28 parts per thousand; median: 24.9 parts per thousand) and quartz (18.0 parts per thousand. to 29.1 parts per thousand; median: 22.6 parts per thousand). Isotopic disequilibrium exists at quartz/wollastonite and wollastonite/calcite boundaries. Therefore, classical batch/Rayleigh fractionation models based on reactant and product equilibrium are not applicable to the wollastonite rims. An approach that relies on local instantaneous mass balance for the reactants, based on the wollastonite-forming reaction is suggested as an alternative way to model wollastonite reaction rims. This model reproduces many of the measured delta O-18 values of wollastonite reaction rims of the current study to within +/- 1 parts per thousand, even though the wollastonite compositions vary by almost 10 parts per thousand. (C) 2011 Elsevier B.V. All rights reserved.

Geological setting of the Guelb Moghrein Fe oxide-Cu-Au-Co mineralization, Akjoujt area, Mauritania

The Guelb Moghrein Fe oxide-Cu-Au-Co deposit is located at the western boundary of the West African craton in NW Mauritania. The wall rocks to the mineralization represent a meta-volcanosedimentary succession typical of Archaean greenstone belts. Two types of meta-volcanic rocks are distinguished: (1) volcanoclastic rocks of rhyodacite-dacite composition (Sainte Barbe volcanic unit), which form the stratigraphic base; (2) tholeiitic andesites-basalts (Akjoujt meta-basalt unit). The trace element signature of both types is characteristic of a volcanic arc setting. A small meta-pelitic division belongs to the Sainte Barbe volcanic unit. A meta-carbonate body, which contains the mineralization, forms a tectonic lens in the Akjoujt meta-basalt unit. It can be defined by the high X(mg) (=36) of Fe-Mg carbonate, the REE pattern and the delta(13)C values of -18 to -17 parts per thousand as a marine precipitate similar to Archaean banded iron formation (BIF). Additionally, small slices of Fe-Mg clinoamphibole-chlorite schist in the meta-carbonate show characteristics of marine shale. This assemblage, therefore, does not represent an alteration product, but represents an iron formation unit deposited on a continental shelf, which probably belongs to the Lembeitih Formation. The hydrothermal mineralization at 2492 Ma was contemporaneous with regional D(2) thrusting of the Sainte Barbe volcanic unit and imbrications of the meta-carbonate in the upper greenschist facies. This resulted in the formation of an ore breccia in the meta-carbonate, which is enriched in Fe, Ni, Co, Cu, Bi, Mo, As and Au. Massive sulphide ore breccia contains up to 20 wt% Cu. The ore fluid was aqueous-carbonic in nature and either changed its composition from a Mg-rich oxidizing to an Fe-rich reducing fluid or the two fluid types mixed at the trap site. All lithologies at Guelb Moghrein were deformed by D(3) thrusting to the east in the lower greenschist facies. The mobility of REE in the retrogressed rocks explains the formation of a second generation of hydrothermal monazite, which was dated at c. 1742 Ma. Archaean rocks of the West African craton extend to the west to Guelb Moghrein. The active continental margin was deformed and mineralized in the Late Archaean-Early Proterozoic and again reactivated in the Mid-Proterozoic and Westphalian, showing that the western boundary of the craton was reactivated several times.

The duration of prograde garnet crystallization in the UHP eclogites at Lago di Cignana, Italy

The distinct core-to-rim zonation of different REEs in garnet in metamorphic rocks, specifically Sm relative to Lu, suggests that Sm-Nd and Lu-Hf isochron ages will record different times along a prograde garnet growth history. Therefore, REE zonations in garnet must be measured in order to correctly interpret the isochron ages in terms of the garnet growth interval, which could span several m.y. New REE profiles, garnet crystal size distributions, and garnet growth modeling, combined with previously published Sm-Nd and Lu-Hf geochronology on a UHP eclogite of the Zermatt-Saas Fee (ZSF) ophiolite, Lago di Cignana (Italy), demonstrate that prograde garnet growth of this sample occurred over a similar to 30 to 40 m.y. interval. Relative to peak metamorphism at 38 to 40 Ma, garnet growth is estimated to have begun at similar to 11 to 14 kbar pressure at similar to 70 to 80 Ma. Although such a protracted garnet growth interval is surprising, this is supported by plate tectonic reconstructions which suggest that subduction of the Liguro-Piemont ocean occurred through slow and oblique convergence. These results demonstrate that REE zonations in garnet, coupled to crystal size distributions, provide a powerful means for understanding prograde metamorphic paths when combined with Sm-Nd and Lu-Hf geochronology. (C) 2009 Elsevier B.V. All rights reserved.

Prolonged survival for patients with newly diagnosed, inoperable glioblastoma with 3-times daily ultrafractionated radiation therapy.

Ultrafractionation of radiation therapy is a novel regimen consisting of irradiating tumors several times daily, delivering low doses (<0.75 Gy) at which hyperradiosensitivity occurs. We recently demonstrated the high efficiency of ultrafractionated radiotherapy (RT) on glioma xenografts and report here on a phase II clinical trial to determine the safety, tolerability, and efficacy of an ultrafractionation regimen in patients with newly and inoperable glioblastoma (GBM). Thirty-one patients with histologically proven, newly diagnosed, and unresectable supratentorial GBM (WHO grade IV) were enrolled. Three daily doses of 0.75 Gy were delivered at least 4 hours apart, 5 days per week over 6-7 consecutive weeks (90 fractions for a total of 67.5 Gy). Conformal irradiation included the tumor bulk with a margin of 2.5 cm. The primary end points were safety, toxicity, and tolerability, and the secondary end points were overall survival (OS) and progression-free survival (PFS). Multivariate analysis was used to compare the OS and PFS with the EORTC-NCIC trial 26981-22981/CE.3 of RT alone vs radiation therapy and temozolomide (TMZ). The ultrafractionation radiation regimen was safe and well tolerated. No acute Grade III and/or IV CNS toxicity was observed. Median PFS and OS from initial diagnosis were 5.1 and 9.5 months, respectively. When comparing with the EORTC/NCIC trial, in both PFS and OS multivariate analysis, ultrafractionation showed superiority over RT alone, but not over RT and TMZ. The ultrafractionation regimen is safe and may prolong the survival of patients with GBM. Further investigation is warranted and a trial associating ultra-fractionation and TMZ is ongoing.

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The stable isotope composition of ostracod fossils is useful for palaeoenvironmental reconstruction. Laboratory as well as 'natural environment' cultures demonstrate that the carbon and oxygen isotope compositions of ostracod shells faithfully record the environmental conditions at the time of valve precipitation. Oxygen isotope composition of ostracod calcite reflects the composition and temperature of the host water, but ostracod shells are enriched in 18O compared to the value expected for inorganic calcite precipitating under equilibrium under the same conditions. This 'vital effect' is generally constant for closely related species and equals 1.5 to 3 0/00 for Candoninae, 0 to 2.5 0/00 for Cyprididae and 0.8 to 1.5 0/00 for Cytheroidea. The carbon isotope composition of ostracod calcite is controlled by a complex interaction between the ecology of the species and environmental parameters. Previous natural environmental studies suggest that many (but not all) taxa crystallize their shells in or very close to equilibrium with the carbon isotope composition of dissolved inorganic carbon, and that the composition of littoral, epifaunal species reflects seasonal variation in the carbon isotope composition of dissolved inorganic carbon of bottom water according to their life cycles, while that of deep-water, infaunal species reflects the variation interstitial pore water according to microhabitat preferences.

Formation of chlorite during thrust fault reactivation. Record of fluid origin and P-T conditions in the Monte Perdido thrust fault (southern Pyrenees)

The chemical and isotopic compositions of clay minerals such as illite and chlorite are commonly used to quantify diagenetic and low-grade metamorphic conditions, an approach that is also used in the present study of the Monte Perdido thrust fault from the South Pyrenean fold-and-thrust belt. The Monte Perdido thrust fault is a shallow thrust juxtaposing upper Cretaceous-Paleocene platform carbonates and Lower Eocene marls and turbidites from the Jaca basin. The core zone of the fault, about 6 m thick, consists of intensely deformed clay-bearing rocks bounded by major shear surfaces. Illite and chlorite are the main hydrous minerals in the fault zone. Illite is oriented along cleavage planes while chlorite formed along shear veins (< 50 mu m in thickness). Authigenic chlorite provides essential information about the origin of fluids and their temperature. delta O-18 and delta D values of newly formed chlorite support equilibration with sedimentary interstitial water, directly derived from the local hanging wall and footwall during deformation. Given the absence of large-scale fluid flow, the mineralization observed in the thrust faults records the P-T conditions of thrust activity. Temperatures of chlorite formation of about 240A degrees C are obtained via two independent methods: chlorite compositional thermometers and oxygen isotope fractionation between cogenetic chlorite and quartz. Burial depth conditions of 7 km are determined for the Monte Perdido thrust reactivation, coupling calculated temperature and fluid inclusion isochores. The present study demonstrates that both isotopic and thermodynamic methods applied to clay minerals formed in thrust fault are useful to help constrain diagenetic and low-grade metamorphic conditions.

Efectos de la aplicación de biochar en el modelo jerárquico de agregación de un suelo forestal bajo condiciones oceánicas

El objetivo del presente proyecto ha sido estudiar los efectos del biochar en el modelo jerárquico de agregación del suelo. Para estudiar estas propiedades se ha realizado una incubación de tres tratamientos de biochar Miscanthus (biochar 10t/ha (B10), 2t/ha (B2) y 10t/ha+Nitrógeno (BN), durante 91 días, realizando fraccionamientos en húmedo de las muestras los días 28, 43, 63, 77 y 91. En estos fraccionamientos se han separado las fracciones macroagregrado (LMagg+Magg), microagregados (magg), limos (Silt) y arcillas (Clay). Por último se determinó el C orgánico de todas las fracciones de los tratamientos obtenidos con el objeto de obtener información sobre el efecto que puede tener la aplicación del biochar sobre la estabilización de C en las fracciones del suelo. Los resultados obtenidos indican que con la aplicación de biochar, la teoría jerárquica de agregación propuesta por Tisdall y Oades (1982), no se cumple en la dinámica de agregación del suelo, si bien se puede observar una jerarquía en la estabilización del C orgánico en los macroagregados y microagregados. Además las dosis altas de biochar producen mayor cantidad de microagregados, favoreciendo el secuestro de C en el suelo. Por otro lado, con dosis altas de biochar se aprecia una disminución de la fracción de macroagregados, hecho que puede influir negativamente en la estructura del suelo.