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Environmental Samples (environmental + sample)
Selected AbstractsPHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE, DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES,JOURNAL OF PHYCOLOGY, Issue 5 2009Sara M. Handy Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis. [source] PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES,JOURNAL OF PHYCOLOGY, Issue 4 2003Ingo Busse Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well-known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group. [source] Automatic Voltammetric System for Continuous Trace Metal Monitoring in Various Environmental SamplesELECTROANALYSIS, Issue 19-20 2007Øyvind Mikkelsen Abstract Some recent developments and results in the field of automatic monitoring of electrolabile concentration of zinc and iron in the low ,g/L range in river water, drainage water, and waste water by use of solid dental amalgam electrode (DAM) as a working electrode are reviewed for three different geographical sites representing the mentioned matrixes. At all sites, voltammetric measurements were carried out continuously every 30 or 60,minutes for periods up to 4,months, and compared with total amounts of the metals found by ICP-MS on manually collected samples. In total, the observed concentration ranges analyzed was in the ranges of sub-,g/L to approximately 30,,g/L for zinc, and from approximately 1,,g/L to 150,,g/L. for iron. Results shows good calibration curves for the metals in the different matrixes (r2avg=0.99) with standard deviation within 5%. The voltammetric system showed good stability and gave reliable results which were in a reasonable agreement with ICP-MS measurements for all analyses when comparing the concentration trends. The frequency of maintenance varied from once a week in waste water samples to once a month in river water. [source] Recent Developments in Trace Element Analysis by ICP-AES and ICP-MS with Particular Reference to Geological and Environmental SamplesGEOSTANDARDS & GEOANALYTICAL RESEARCH, Issue 1 2005Kathryn L. Linge This review describes recent developments in trace element analysis using inductively coupled plasma-atomic emission spectrometry (ICP-AES) and inductively coupled plasma-mass spectrometry (ICP-MS). It aims to focus on the application of ICP techniques to geological and environmental samples. Therefore, fundamental studies in ICP-MS and ICP-AES instrumentation have largely been ignored. Whereas the majority of literature reviewed related to ICP-MS, indicating that ICP-MS is now the preferred technique for all geological analysis, there is still a steady development of ICP-AES to environmental applications. It is clear that true flexibility in elemental analysis can only be achieved by combining the advantages of both ICP-AES and ICP-MS. Two particular groups of elements (long-lived radionuclide and the platinum-group elements) stood out as warranting dedicated sections describing analytical developments these areas. [source] Facile and Sensitive Spectrophotometric Determination of Propoxur in Formulations and Environmental SamplesHELVETICA CHIMICA ACTA, Issue 5 2005Kailasa, Suresh Kumar A facile, rapid, and sensitive spectrophotometric method for the determination of propoxur in insecticidal formulations, fortified water, vegetables, agricultural wastewater, and agricultural soil samples has been elaborated. The proposed method is based on the hydrolysis of propoxur under basic conditions, followed by instantaneous azo coupling of the resulting 2-isopropoxyphenol with the anilines 2a,c. This yielded the orange-red chromophore 3a (,max=at 470,nm), the pale-red coupling product 3b (490,nm), or the red derivative 3c (478,nm), which are stable for 46,h, 38,h, and 24,h, respectively, and could be readily analyzed spectrophotometrically. [source] Evaluating polycyclic aromatic hydrocarbons using a yeast bioassayENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2007Abeer Alnafisi Abstract Sixteen polycyclic aromatic hydrocarbons (PAHs) were evaluated for the ability to activate aryl hydrocarbon (Ah) receptor signaling in a yeast-based bioassay. Individual PAHs were classified as inactive or as weakly, moderately, or strongly active based on induction of human Ah receptor signaling. Indeno[1,2,3- cd]pyrene, chrysene, benzo[a]anthracene, benzo[a]pyrene, benzo[j]fluoranthene, and benzo[k]fluoranthene were the most potent activators of human Ah receptor signaling. Various mixtures of PAHs had additive or synergistic effects in the bioassay. Environmental samples from the New Orleans (Louisiana, USA) and Detroit (Michigan, USA) areas that were previously analyzed for PAH composition and quantity were tested in this bioassay. Weak but statistically significant relationships were found when the analytically measured levels of PAHs were correlated with sample dilutions that gave 25% effective concentration signaling levels in the Ah receptor assay. We conclude that this Ah receptor signaling assay may be useful for preliminary biomonitoring of samples for PAHs and other Ah receptor ligands. [source] Microbial community analysis at crude oil-contaminated soils targeting the 16S ribosomal RNA, xylM, C23O, and bcr genesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009Y. Higashioka Abstract Aims:, The analyses targeting multiple functional genes were performed on the samples of crude oil-contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Methods and Results:, Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3-dioxygenase (C23O), and benzoyl-CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. Conclusions:, The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. Significance and Impact of the Study:, The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil-contaminated environments. [source] Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutantsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Jisung Ryu Abstract The sequence of cytochrome P4501A (CYP1A) cDNA of medaka (Oryzias latipes) was determined, and its messenger ribonucleic acid (mRNA) regulation by ,-naphthoflavone (,NF) was evaluated. The determined cDNA sequence contained 2,349 base pairs (bp), and the open reading frame contained a total of 1,563 bp encoding 521 predicted amino acids. The induction of CYP1A mRNA in medaka was evaluated using reverse transcription,polymerase chain reaction. The concentration,dependent induction of CYP1A mRNA in the liver was observed after exposure to ,NF at nominal concentrations of 20, 100, and 500 ,g/ L for 2 d. Time-dependent changes of CYP1A mRNA levels were also observed in the liver, gill, gut, and caudal fin tissues of medaka exposed to 100 ,g/L of ,NF for 7 d. Our results showed that the degree of CYP1A mRNA induction in the gill, gut, and caudal fin after exposure to ,NF was relatively higher than that in the liver, possibly because of low basal levels of CYP1A mRNA in the gill, gut, and caudal fin of nonexposed fish. The induction of medaka CYP1A mRNA was also observed after exposure to an environmental sample, landfill leachate. The CYP1A mRNA inductions in the gill, gut, and caudal fin were also higher than that in the liver as shown in the ,NF-treated groups. These results show that CYP1A mRNA determination in the gill, gut, and caudal fin, which are in direct contact with the polluted water, may become a useful method for monitoring CYP1A-inducible chemicals. [source] Sponge halogenated natural products found at parts-per-million levels in marine mammalsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2002Walter Vetter Abstract Several unknown, abundant brominated compounds (BCs) were recently detected in the blubber of dolphins and other marine mammals from Queensland (northeast Australia). The BCs were interpreted as potential natural products due to the lack of anthropogenic sources for these compounds. This study investigated whether some of the BCs accumulated by diverse marine mammal species are identical with natural BCs previously isolated from sponges (Dysidea sp.) living in the same habitat. Isolates from sponges and mollusks (Asteronotus cespitosus) were compared with the signals detected in the mammals' tissue. Mass spectra and gas chromatography retention times on four different capillary columns of the isolates from sponges and mammals were identical in all respects. This proves that the chemical name of the compound previously labeled BC-2 is 4,6-dibromo-2-(2,,4,-dibromo)phenoxyanisole and that the chemical name of BC-11 is 3,5-dibromo-2-(3,,5,-dibromo,2,-methoxy)phenoxyanisole. Using a quantitative reference solution of BC-2, we established that the concentrations of the brominated metabolites found in the marine mammals are frequently >1 mg/kg. The highest concentration (3.8 mg/kg), found in a sample of pygmy sperm whale (Kogia breviceps), indicates that BC-2 is a bioaccumulative, natural organohalogen compound. This is supported by the concentrations of the BCs in our samples being equal to the highest concentrations of anthropogenic BCs in any environmental sample. The quantitative determination of BC-2 in blubber of marine mammals from Africa and the Antarctic suggests that BC-2 is widespread. These results are direct proof that marine biota can produce persistent organic chemicals that accumulate to substantial concentrations in higher trophic organisms. [source] New directions and interactions in metagenomics researchFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Naomi Ward Abstract Metagenomics, which aims to access the genomic potential of an environmental sample directly, is a burgeoning area that is generating enormous amounts of biological information. An examination of recent metagenomics literature reveals the discipline to be heading in new and interesting directions, including the investigation of the normal flora of mammals, analysis of ancient genomes, and exploration of the distribution of novel pathways. In addition, the development of new bioinformatics approaches and tools is allowing innovative mining of both existing and new data. Finally, there are indications that the integration of metagenomics with complementary approaches in microbial ecology is beginning. [source] Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGEFEMS MICROBIOLOGY ECOLOGY, Issue 3 2005Dorthe Groth Petersen Abstract The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCRIS) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCRIS during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel. [source] Rapid preparation of cyanobacterial DNA for real-time PCR analysisLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2008J.P. Rasmussen Abstract Aims:, To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. Methods and Results:, Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. Conclusions:, Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. Significance and Impact of the Study:, The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices. [source] Electrochemical Detection of Arsenic(III) in the Presence of Dissolved Organic Matter (DOM) by Adsorptive Square-Wave Cathodic Stripping Voltammetry (Ad-SWCSV)ELECTROANALYSIS, Issue 4 2008Tsanangurayi Tongesayi Abstract This study has demonstrated that As(III) can be electrochemically detected and quantified in the presence of fulvic acid (FA) and dissolved organic matter (DOM). This eliminates the need to remove DOM prior to measurement of As(III) in environmental samples. Apart from reducing analysis time and the cost of the analysis, this could be potentially useful for the development of electrochemical methods for the detection and measurement of As(III) onsite. Both synthetic samples in which FA was added and a real sample with 22.16,mg/L total organic carbon (TOC) were analyzed. [source] Peptide Modified Electrodes as Electrochemical Metal Ion SensorsELECTROANALYSIS, Issue 15 2006Edith Chow Abstract Sensors for the detection of metal ions are of considerable importance for enabling the monitoring of environmental samples for metal ion contamination directly in the field. This review outlines the use of peptides and amino acids as the recognition element of electrochemical sensors for metal ion detection. Initially the complexation of metals by peptides is discussed followed by the immobilization of peptides on electrode surfaces. Subsequently, the application of peptide modified electrodes for detecting metals is reviewed and finally challenges and future prospects are outlined. [source] Catalytic Adsorptive Stripping Voltammetric Procedure for Determination of Total Chromium in Environmental MaterialsELECTROANALYSIS, Issue 12 2006gorzata Grabarczyk Abstract A sensitive catalytic adsorptive stripping voltammetric procedure for determination of traces of total chromium in environmental samples is reported. The method is based on the preconcentration of a Cr(III)H2DTPA complex by adsorption at the HMDE from an acetate buffer solution at the potential ,1.0,V vs. Ag/AgCl. Total chromium was determined as Cr(III) after reduction of Cr(VI) to Cr(III) by NaHSO3. In order to stabilize the signal of Cr(III) the measurements were performed at 5,°C. The calibration graph for chromium for an accumulation time of 60,s was linear in the range from 5×10,10 to 5×10,8,mol L,1. The relative standard deviation for a chromium concentration of 1×10,8,mol L,1 was 3.9% (n=5). The detection limit for accumulation time of 60,s was about 8×10,11,mol L,1. The validation of the procedure was performed by the analysis of the certified reference materials. [source] Development of a Rapid Single-Drop Analysis Biosensor for Screening of Phenanthrene in Water SamplesELECTROANALYSIS, Issue 20 2004Abstract Detection techniques for biosensors often require bulky instruments or cells that are not feasible for in-field analysis. Our single-drop cell design, optimized in this work, comprised a screen-printed three-electrode (SPE), strip in horizontal position onto which a volume of 100,,L of sample or substrate solution was placed to ensure electrical contact (complete circuit). Together with optimized linear sweep voltammetry (LSV), parameters for the detection of the enzyme alkaline phosphatase (AP), the system was applied to a biosensor for the analysis of polycyclic aromatic hydrocarbons (PAHs), in environmental samples. A limit of detection (LOD), of 0.15,ppb was achieved for a model system with an IC50 value of 0.885 ppb and a linear range (LR), of 0.2,10,ppb. Application of the single drop analysis (SDA), format to a PAH biosensor gave a LOD of 1.4,ppb for detection of phenanthrene with an IC50 value of 29.3,ppb and linear range of 2,100,ppb. Proof of concept is shown with spiked sample analysis of phenanthrene in matrices such as sea, river and tap water. [source] Cover Picture: Electrophoresis 15/2008ELECTROPHORESIS, Issue 15 2008Article first published online: 24 JUL 200 Regular issues provide a wide range of research and review articles covering all aspects of electrophoresis. Here you will find cutting-edge articles on methods and theory, instrumentation, nucleic acids, CE and CEC, miniaturization and microfluidics, proteomics and two-dimensional electrophoresis. Selected topics of issue 15 are: The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis Metabolic fingerprinting of schistosoma mansoni infection in mice urine with capillary electrophoresis Supercritical fluid extraction as an on-line clean-up technique for determination of riboflavin vitamins in food samples by capillary electrophoresis with fluorimetric detection A two-step electro-dialysis method for DNA purification from polluted metallic environmental samples. [source] "In-gel patch electrophoresis:" A,new method for environmental DNA purificationELECTROPHORESIS, Issue 16 2005Changhyun Roh Abstract Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications. [source] Modified Hadamard transform microchip electrophoresisELECTROPHORESIS, Issue 16 2005Renato Guchardi Abstract Sensitivity is a crucial point in the development applications for medicine or environmental samples in which the analytes are present in the nanomolar range. Besides further technical development of detection systems, the multiplex sample injection technique can be applied for enhancing the signal-to-noise ratio. Hadamard transform is easily applied to microchip electrophoresis due to the fact that sample injection is generally achieved through cross, double-tee, or tee injector structures. This paper reports the first demonstration of a modified Hadamard transform electrophoresis on a microchip by using an amperometric detector. Contrary to the previous Hadamard applications, the resolution (number of points per unit of time) of electropherograms obtained is independent of the number of injections. [source] Use of a high-throughput umu -microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matterENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2004Yoshimitsu Oda Abstract In the present study, we developed a rapid umu -microplate test system that uses the nitroreductase- and O -acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O -acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative ,-galactosidase activities were then determined colorimetrically using either chlorophenol red-,- D -galactopyranoside (CPRG) or O -nitrophenyl-,- D -galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu -microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu -microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples. Environ. Mol. Mutagen. 43:10,19, 2004. © 2004 Wiley-Liss, Inc. [source] Eukaryotic diversity and phylogeny using small- and large-subunit ribosomal RNA genes from environmental samplesENVIRONMENTAL MICROBIOLOGY, Issue 12 2009William Marande Summary The recent introduction of molecular techniques in eukaryotic microbial diversity studies, in particular those based in the amplification and sequencing of small-subunit ribosomal DNA (SSU rDNA), has revealed the existence of an unexpected variety of new phylotypes. The taxonomic ascription of the organisms bearing those sequences is generally deduced from phylogenetic analysis. Unfortunately, the SSU rDNA sequence alone has often not enough phylogenetic information to resolve the phylogeny of fast-evolving or very divergent sequences, leading to their misclassification. To address this problem, we tried to increase the phylogenetic signal by amplifying the complete eukaryotic rDNA cluster [i.e. the SSU rDNA, the internal transcribed spacers, the 5.8S rDNA and the large-subunit (LSU) rDNA] from environmental samples, and sequencing the SSU and LSU rDNA part of the clones. Using marine planktonic samples, we showed that surveys based on either SSU or SSU + LSU rDNA retrieved comparable diversity patterns. In addition, phylogenetic trees based on the concatenated SSU + LSU rDNA sequences showed better resolution, yielding good support for major eukaryotic groups such as the Opisthokonta, Rhizaria and Excavata. Finally, highly divergent SSU rDNA sequences, whose phylogenetic position was impossible to determine with the SSU rDNA data alone, could be placed correctly with the SSU + LSU rDNA approach. These results suggest that this method can be useful, in particular for the analysis of eukaryotic microbial communities rich in phylotypes of difficult phylogenetic ascription. [source] Culture-independent evidence for the persistent presence and genetic diversity of microcystin-producing Anabaena (Cyanobacteria) in the Gulf of FinlandENVIRONMENTAL MICROBIOLOGY, Issue 4 2009David P. Fewer Summary The late summer mass occurrences of cyanobacteria in the Baltic Sea are among the largest in the world. These blooms are rarely monotypic and are often composed of a diverse assemblage of cyanobacteria. The toxicity of the blooms is attributed to Nodularia spumigena through the production of the hepatotoxic nodularin. However, the microcystin hepatotoxins have also been reported from the Baltic Sea on a number of occasions. Recent evidence links microcystin production in the Gulf of Finland directly to the genus Anabaena. Here we developed a denaturing gradient gel electrophoresis (DGGE) method based on the mcyE microcystin synthetase gene and ndaF nodularin synthetase gene that allows the culture-independent discrimination of microcystin- and nodularin-producing cyanobacteria directly from environmental samples. We PCR-amplified microcystin and nodularin synthetase genes from environmental samples taken from the Gulf of Finland and separated them on a denaturing gradient gel using optimized conditions. Sequence analyses demonstrate that uncultured microcystin-producing Anabaena strains are genetically more diverse than previously demonstrated from cultured strains. Furthermore, our data show that microcystin-producing Anabaena are widespread in the open Gulf of Finland. Non-parametric statistical analysis suggested that salinity plays an important role in defining the distribution of microcystin-producing Anabaena. Our results indicate that microcystin-producing blooms are a persistent phenomenon in the Gulf of Finland. [source] Amplification of low quantity bacterial RNA for microarray studies: time-course analysis of Leptospirillum ferrooxidans under nitrogen-fixing conditions,ENVIRONMENTAL MICROBIOLOGY, Issue 6 2006Mercedes Moreno-Paz Summary We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3, end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130,150 µg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram-negative bacterium Leptospirillum ferrooxidans. We further validated our amplification method in parallel experiments with non-amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time-course of growth. [source] DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 2 2006Gian Marco Luna Summary In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H,) and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of ,-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysisENVIRONMENTAL TOXICOLOGY, Issue 6 2001Judith A. Baker Abstract We report molecular analyses which identify cyanobacterial strains present in environmental samples. These analyses do not require the isolation and culture of strains. Identification of cyanobacteria used the polymerase chain reaction (PCR), based on the phycocyanin operon. Differentiation was either by restriction endonuclease digestion (restriction fragment length polymorphisms) or sequencing of the PCR products. Identification was based on sequence homology of the intergenic spacer region (IGS) between the ,- and ,-phycocyanin subunits (PC-IGS) with database records. We have found that the length and sequence of the PC-IGS is capable of predicting the genus accurately, but not the species. Toxigenicity was determined with oligonucleotide probes for key steps in the microcystin toxin synthesis pathway. We have shown that it is possible to easily and routinely obtain PCR amplification products and differentiate the strains in bloom samples. The methods can detect even minor components in bloom samples, which may not be apparent on microscopic examination. Genetic probes for microcystin toxigenicity are effective on environmental samples, eliminating the need for isolation and culture of the organisms. The use of a suite of tests described here will allow water managers to determine the presence and the type of cyanobacteria and their microcystin toxigenicity. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 472,482, 2001 [source] Comparative sensitivity of embryo,larval toxicity assays with African catfish (Clarias gariepinus) and zebra fish (Danio rerio)ENVIRONMENTAL TOXICOLOGY, Issue 6 2001Lien T. H. Nguyen Abstract Embryo,larval toxicity tests with the African catfish (Clarias gariepinus) were conducted with five chemicals (Cr, Cd, Zn, NaPCP and malathion) and three environmental samples. The sensitivity of the 5-day assay was compared to that of the 12-day embryo,larval toxicity tests with the zebra fish (Danio rerio). The ratios of the C. gariepinus and D. rerio LC50 values ranged from 0.4 for Cr to 8.9 for Zn. The ratios of subchronic values ranged from 0.25 for NaPCP to 3.1 for Cd indicating a more comparable sensitivity of the two species. For the three sediment pore waters, the ratios were 0.6, 1.1, and 2.4 and the subchronic values were identical for the two species. The results suggest that, considering the short-test duration and its sensitivity, the 5-day embryo,larval tests with C. gariepinus may be a potential alternative for short-term embryo,larval toxicity testing with fish. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 566,571, 2001 [source] Pharmaceutical metabolites in the environment: Analytical challenges and ecological risks,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2009Mary D. Celiz Abstract The occurrence of human and veterinary pharmaceuticals in the environment has been a subject of concern for the past decade because many of these emerging contaminants have been shown to persist in soil and water. Although recent studies indicate that pharmaceutical contaminants can pose long-term ecological risks, many of the investigations regarding risk assessment have only considered the ecotoxicity of the parent drug, with very little attention given to the potential contributions that metabolites may have. The scarcity of available environmental data on the human metabolites excreted into the environment or the microbial metabolites formed during environmental biodegradation of pharmaceutical residues can be attributed to the difficulty in analyzing trace amounts of previously unknown compounds in complex sample matrices. However, with the advent of highly sensitive and powerful analytical instrumentations that have become available commercially, it is likely that an increased number of pharmaceutical metabolites will be identified and included in environmental risk assessment. The present study will present a critical review of available literature on pharmaceutical metabolites, primarily focusing on their analysis and toxicological significance. It is also intended to provide an overview on the recent advances in analytical tools and strategies to facilitate metabolite identification in environmental samples. This review aims to provide insight on what future directions might be taken to help scientists in this challenging task of enhancing the available data on the fate, behavior, and ecotoxicity of pharmaceutical metabolites in the environment. [source] The role of biomarkers to assess oil-contaminated sediment quality using toxicity tests with clams and crabs,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2008Carmen Morales-Caselles Abstract A 28-d bioassay was conducted with two invertebrate species with different feeding habits, the clam Ruditapes philippinarum and the shore crab Carcinus maenas. The purpose of the present study was to assess the quality of sediments affected by oil spills in different areas of the Spanish coast. The organisms were exposed to environmental samples of oil-contaminated sediments during four weeks and, after the experiment, a suite of biomarkers of exposure was measured: The phase one detoxification system was assessed by ethoxyresorufin- O -deethylase (EROD) activity; glutathione- S -transferase (GST) is a phase-two detoxification enzyme but also is implicated in oxidative stress events; glutathione peroxidase (GPX), glutathione reductase (GR), and the ferric reducing ability of plasma (FRAP) assay were analyzed to determine the antioxidant activity of the tissues. The biomarker results were correlated with the chemical compounds bound to sediments (polycyclic aromatic hydrocarbons [PAHs], polychlorinated biphenyls [PCBs], Zn, Cd, Pb, Cu, Ni, Co, V) and a principal component analysis was carried out with the purpose of linking all the variables and to detect those contaminated sediments potentially harmful to the biota. Results showed induction of biomarkers in both invertebrate species and significant differences (p < 0.05; p < 0.01) were established among sediments affected by different spills. The use of the selected biomarkers together with the sediment chemical analysis assesses the bioavailability of contaminants and has proven to be a suitable tool to monitor the environmental quality of sediments affected by oil spills. [source] Toxicological characterization of 2,4,6-trinitrotoluene, its transformation products, and two nitramine explosivesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2007Judith Neuwoehner Abstract The soil and groundwater of former ordnance plants and their dumping sites have often been highly contaminated with the explosive 2,4,6-trinitrotoluene (2,4,6-TNT) leading to a potential hazard for humans and the environment. Further hazards can arise from metabolites of transformation, by-products of the manufacturing process, or incomplete combustion. This work examines the toxicity of polar nitro compounds relative to their parent compound 2,4,6-TNT using four different ecotoxicological bioassays (algae growth inhibition test, daphnids immobilization test, luminescence inhibition test, and cell growth inhibition test), three genotoxicological assays (umu test, NM2009 test, and SOS Chromotest), and the Ames fluctuation test for detection of mutagenicity. For this study, substances typical for certain steps of degradation/transformation of 2,4,6-TNT were chosen for investigation. This work determines that the parent compounds 2,4,6-TNT and 1,3,5-trinitrobenzene are the most toxic substances followed by 3,5-dinitrophenol, 3,5-dinitroaniline and 4-amino-2-nitrotoluene. Less toxic are the direct degradation products of 2,4,6-TNT like 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene. A weak toxic potential was observed for 2,4,6-trinitrobenzoic acid, 2,4-diamino-6-nitrotoluene, 2,4-dinitrotoluene-5-sulfonic acid, and 2,6-diamino-4-nitrotoluene. Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine and hexahydro-1,3,5-trinitro-1,3,5-triazine show no hint of acute toxicity. Based on the results of this study, we recommend expanding future monitoring programs of not only the parent substances but also potential metabolites based on conditions at the contaminated sites and to use bioassays as tools for estimating the toxicological potential directly by testing environmental samples. Site-specific protocols should be developed. If hazardous substances are found in relevant concentrations, action should be taken to prevent potential risks for humans and the environment. Analyses can then be used to prioritise reliable estimates of risk. [source] | ||||||||||||||||||||||||||||||||||