A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS

A simple method for mercury speciation in hair samples with a fast sample preparation procedure using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry is proposed. Prior to analysis, 50 mg of hair samples were accurately weighed into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of inorganic mercury (Ino-Hg), methylmercury (Met-Hg) and ethylmercury (Et-Hg) was accomplished in less than 8 min on a C18 reverse phase column with a mobile phase containing 0.05% v/v mercaptoethanol, 0.4% m/v L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% v/v methanol. The method detection limits were found to be 15 ng g(-1), 10 ng g(-1) and 38 ng g(-1), for inorganic mercury, methylmercury and ethylmercury, respectively. Sample throughput is 4 samples h(-1) (duplicate). A considerable improvement in the time of analysis was achieved when compared to other published methods. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Finally, the proposed method was successfully applied to the speciation of mercury in hair samples collected from fish-eating communities of the Brazilian Amazon.

Evaluation of Antigenotoxic Effects of Plant Flavonoids Quercetin and Rutin on HepG2 Cells

The flavonoid quercetin and its derivative rutin were investigated for genotoxicity/antigenotoxicity activity in human hepatoma HepG2 cells using the comet assay. The extract cytotoxicity was evaluated using the trypan blue exclusion dye method with quercetin and rutin concentrations ranging from 0.1 to 200.0 mu g/mL of culture medium. Three minor non-cytotoxic concentrations were chosen to evaluate the genotoxicity and antigenotoxicity of the flavonoids (0.1, 1.0 and 5.0 mu g/mL) through comet assay. The cultures were treated with three different concentrations of rutin or quercetin (genotoxicity) or their association with Aflatoxin B1 (AFB1), methyl methanesulfonate (MMS) or doxorubicin (DXR) (antigenotoxicity test) in three protocols: pretreatment, simultaneous treatment and post-treatment. The cell cultures were also treated with 1% DMSO (control group), AFB1, MMS and DXR (positive-control). Statistical analyses were performed using ANOVA and Dunnett`s test (p <= 0.05). Quercetin at concentrations higher than 10.0 mu g/mL or rutin higher than 50.0 mu g/mL exhibited a cytotoxic effect on the cells, showing that quercetin is more cytotoxic than rutin. Furthermore, neither compound was able to induce genotoxicity in the concentrations evaluated. On the other hand, both flavonoids reduced DNA damage induced by AFB1, MMS and DXR in all treatment protocols. Copyright (C) 2011 John Wiley & Sons, Ltd.

Determination of total and inorganic mercury in whole blood by cold vapor inductively coupled plasma mass spectrometry (CV ICP-MS) with alkaline sample preparation

A simple method with a fast sample preparation procedure for total and inorganic mercury determinations in blood samples is proposed based on flow injection cold vapor inductively coupled plasma mass spectrometry (FI-CVICP-MS). Aliquots of whole blood (500 mL) are diluted 1 + 1 v/v with 10.0% v/v tetramethylammonium hydroxide (TMAH) solution, incubated for 3 h at room temperature and then further diluted 1 + 4 v/v with 2.0% v/v HCl. The inorganic Hg was released by online addition of L-cysteine and then reduced to elemental Hg by SnCl(2). On the other hand, total mercury was determined by on-line addition of KMnO(4) and then reduced to elemental Hg by NaBH(4). Samples were calibrated against matrix-matching. The method detection limit was found to be 0.80 mu g L(-1) and 0.08 mu g L(-1) for inorganic and total mercury, respectively. Sample throughput is 20 samples h(-1). The method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). For additional validation purposes, human whole blood samples were analyzed by the proposed method and by an established CV AAS method, with no statistical difference between the two techniques at 95% confidence level on applying the t-test.

Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

Screening of plant extracts from the Brazilian Cerrado for their in vitro trypanocidal activity

In this study we report the screening of the in vitro trypanocidal activity of 20 extracts obtained from 10 different plant species growing in the Brazilian Cerrado: Aspidosperma macrocarpum Mart. (Apocynaceae), Aegiphila sellowiano Cham. (Verbenaceae), Byrsonima intermedia Juss. (Malpighiaceae), Cyperus rotundus L. (Cyperaceae), Leandra lacunosa Cogn. (Melastomataceae), Miconia ligustroides (DC.) Naudin. (Melastomataceae), Miconia sellowiana Naudin.(Melastomataceae),Myrcia variabilis Mart.ex DC. (Myrtaceae), Solanum lycocarpum St. Hil. (Solanaceae), and Tibouchina stenocarpa Cogn. (Melastomataceae). The most active extracts were submitted to phytochemical analyses. High-resolution gas chromatography analysis of the n-hexane extract of T. stenocarpa (IC(50) = 23.6 mu g/mL), the most active extract amongst all the tested samples, allowed the identification of beta-amyrin, alpha-amyrin, lupeol, friedelin, beta-friedelanol, campesterol, stigmasterol, and beta-sitosterol. Oleanolic and ursolic acids were isolated from the methylene chloride extract of T stenocarpa (IC(50) = 51.5 mu g/mL), while ursolic acid was isolated from the methylene chloride extract of M. variabilis (IC(50)=38.4 mu g/mL). Solasonine and solamargine were identified as major compounds by mass spectrometry analysis in the hydroalcoholic extract of the fruits of S. lycocarpum (IC(50)=57.1 mu g/mL).The results showed that the trypanocidal activity may be related to the major compounds identified in the crude active extracts.

Bioactive Chemical Constituents and Comparative Antimicrobial Activity of Callus Culture and Adult Plant Extracts from Alternanthera tenella

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirao Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 mu g/mL.

Functionalization of (2S)-Isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones by a Suzuki-Miyaura Cross-Coupling Reaction Using Aryltrifluoroborate Salts: Convenient Enantioselective Preparation of alpha-Substituted beta-Amino Acids

A simple protocol for the Pd(OAc)(2)-catalyzed cross-coupling reaction of 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones with potassium aryltrifluoroborates was developed. The reaction is performed at 110 degrees C with a ligand-free catalyst. In all cases, complete conversion of the 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones and aryltrifluoroborates into the C-C coupling products was observed within 30-360 min. It is noteworthy that a large variety of groups present in the potassium aryltrifluoroborates (-CF(3), -OMe, -SEt, -CN, -CHO, -Cl, -Cbz, -NCbz, -OH, -CO(2)H) could be tolerated. Hydrogenation of the endocyclic double bonds in the Suzuki-Miyaura products followed by acid hydrolysis afforded highly enantioenriched alpha-aryl-substituted beta-amino acids.

Preparation, characterization and in vitro cytotoxicity of BSA-based nanospheres containing nanosized magnetic particles and/or photosensitizer

This study reports on the preparation, characterization and in vitro toxicity test of a new nano-drug delivery system (NDDS) based on bovine serum albumin (BSA) nanospheres which incorporates surface-functionalized magnetic nanoparticles (MNP) and/or the silicon(IV) phthalocyanine (NzPc). The new NDDS was engineered for use in photodynamic therapy (PDT) combined with hyperthermia (HPT) to address cancer treatment. The BSA-based nanospheres, hosting NzPc, MNP or both (NzPc and MNP), present spherical shape with hydrodynamic average diameter values ranging from 170 to 450 nm and zeta potential of around -23 mV. No difference on the fluorescence spectrum of the encapsulated NzPc was found regardless of the presence of MNP. Time-dependent fluorescence measurements of the encapsulated NzPc revealed a bi-exponential decay for samples incorporating only NzPc and NzPc plus MNP, in the time window ranging from 1.70 to 5.20 ns. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic NDDS. (C) 2009 Elsevier B.V. All rights reserved.

Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: An in vitro analysis

The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 mu g/mL, M. cabucu up to 40 mu g/mL, M. albicans up to 40 mu g/mL and M. stenostachya up to 60 mu g/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 mu g/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity. (C) 2010 Elsevier GmbH. All rights reserved.

CO2 reforming of methane on Ni catalysts: Effects of the support phase and preparation technique

The effects of the support phase and catalyst preparation methods on catalytic activity and carbon deposition were systematically investigated over nickel catalysts supported on Al2O3, SiO2 and MgO for the reforming reaction of methane with carbon dioxide. It is found that the pore structure of the support and metal-support interaction significantly affected the catalytic activity and coking resistance. Catalyst with well-developed porosity exhibited higher catalytic activity. Strong interaction between metal and the support made the catalyst more resistant to sintering and coking, thus resulting in a longer time of catalyst stability. (C) 1998 Elsevier Science B.V.

Measurement and state preparation via ion trap quantum computing

We investigate in detail the effects of a QND vibrational number measurement made on single ions in a recently proposed measurement scheme for the vibrational state of a register of ions in a linear rf trap [C. D'HELON and G. J. MILBURN, Phys Rev. A 54, 5141 (1996)]. The performance of a measurement shows some interesting patterns which are closely related to searching.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.

Preparation, characterization, and catalytic properties of clay-based nickel catalysts for methane reforming

Naturally occurring clays and pillared clays are used as supports of nickel catalysts for the methane reforming reaction with carbon dioxide to synthesis gas. The structural and textural characteristics of the supports and catalysts are systematically examined by N-2 adsorption/desorption and X-ray diffraction (XRD) techniques. It is found that the pore structures and surface properties of supports greatly affect the catalytic activities of the catalysts prepared. The catalysts supported on the mesoporous clays or pillared clays are obviously superior to those on microporous supports because the mesoporous supports are highly thermal stable compared to the microporous ones. It is found that introducing lanthanum to the supports can improve the catalyst basicity and thus enhance the catalytic activities of these catalysts. Deactivation of catalysts prepared and factors influencing their stability are also discussed. (C) 1998 Academic Press.

Nitrogen relations of natural and disturbed plant communities in tropical Australia

Nitrogen relations of natural and disturbed tropical plant communities in northern Australia (Kakadu National Park) were studied. Plant and soil N characteristics suggested that differences in N source utilisation occur at community and species level. Leaf and xylem sap N concentrations of plants in different communities were correlated with the availability of inorganic soil N (NH4+ and NO3-). In general, rates of leaf NO3- assimilation were low. Even in communities with a higher N status, including deciduous monsoon forest, disturbed wetland, and a revegetated mine waste rock dump, levels of leaf nitrate reductase, xylem and leaf NO3 levels were considerably lower than those that have been reported for eutrophic communities. Although NO3- assimilation in escarpment and eucalypt woodlands, and wetland, was generally low, within these communities there was a suite of species that exhibited a greater capacity for NO3- assimilation. These high-NO3- species were mainly annuals, resprouting herbs or deciduous trees that had leaves with high N contents. Ficus, a high-NO3- species, was associated with soil exhibiting higher rates of net mineralisation and net nitrification. Low-NO3- species were evergreen perennials with low leaf N concentrations. A third group of plants, which assimilated NO3- (albeit at lower rates than the high-NO3- species), and had high-N leaves, were leguminous species. Acacia species, common in woodlands, had the highest leaf N contents of all woody species. Acacia species appeared to have the greatest potential to utilise the entire spectrum of available N sources. This versatility in N source utilisation may be important in relation to their high tissue N status and comparatively short life cycle. Differences in N utilisation are discussed in the context of species life strategies and mycorrhizal associations.

Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli

MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 with an additional ATG: start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coil extract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coil. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens. (C) 1999 Academic Press.