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Sample records for archaeal orfeomes subcellular

  1. CoBaltDB: Complete bacterial and archaeal orfeomes subcellular localization database and associated resources

    Directory of Open Access Journals (Sweden)

    Lucchetti-Miganeh Céline

    2010-03-01

    Full Text Available Abstract Background The functions of proteins are strongly related to their localization in cell compartments (for example the cytoplasm or membranes but the experimental determination of the sub-cellular localization of proteomes is laborious and expensive. A fast and low-cost alternative approach is in silico prediction, based on features of the protein primary sequences. However, biologists are confronted with a very large number of computational tools that use different methods that address various localization features with diverse specificities and sensitivities. As a result, exploiting these computer resources to predict protein localization accurately involves querying all tools and comparing every prediction output; this is a painstaking task. Therefore, we developed a comprehensive database, called CoBaltDB, that gathers all prediction outputs concerning complete prokaryotic proteomes. Description The current version of CoBaltDB integrates the results of 43 localization predictors for 784 complete bacterial and archaeal proteomes (2.548.292 proteins in total. CoBaltDB supplies a simple user-friendly interface for retrieving and exploring relevant information about predicted features (such as signal peptide cleavage sites and transmembrane segments. Data are organized into three work-sets ("specialized tools", "meta-tools" and "additional tools". The database can be queried using the organism name, a locus tag or a list of locus tags and may be browsed using numerous graphical and text displays. Conclusions With its new functionalities, CoBaltDB is a novel powerful platform that provides easy access to the results of multiple localization tools and support for predicting prokaryotic protein localizations with higher confidence than previously possible. CoBaltDB is available at http://www.umr6026.univ-rennes1.fr/english/home/research/basic/software/cobalten.

  2. The Xenopus ORFeome: A resource that enables functional genomics.

    Science.gov (United States)

    Grant, Ian M; Balcha, Dawit; Hao, Tong; Shen, Yun; Trivedi, Prasad; Patrushev, Ilya; Fortriede, Joshua D; Karpinka, John B; Liu, Limin; Zorn, Aaron M; Stukenberg, P Todd; Hill, David E; Gilchrist, Michael J

    2015-12-15

    Functional characterisation of proteins and large-scale, systems-level studies are enabled by extensive sets of cloned open reading frames (ORFs) in an easily-accessible format that enables many different applications. Here we report the release of the first stage of the Xenopus ORFeome, which contains 8673 ORFs from the Xenopus Gene Collection (XGC) for Xenopus laevis, cloned into a Gateway® donor vector enabling rapid in-frame transfer of the ORFs to expression vectors. This resource represents an estimated 7871 unique genes, approximately 40% of the non-redundant X. laevis gene complement, and includes 2724 genes where the human ortholog has an association with disease. Transfer into the Gateway system was validated by 5' and 3' end sequencing of the entire collection and protein expression of a set of test clones. In a parallel process, the underlying ORF predictions from the original XGC collection were re-analysed to verify quality and full-length status, identifying those proteins likely to exhibit truncations when translated. These data are integrated into Xenbase, the Xenopus community database, which associates genomic, expression, function and human disease model metadata to each ORF, enabling end-users to search for ORFeome clones with links to commercial distributors of the collection. When coupled with the experimental advantages of Xenopus eggs and embryos, the ORFeome collection represents a valuable resource for functional genomics and disease modelling. PMID:26391338

  3. ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

    Science.gov (United States)

    Pellet, J; Tafforeau, L; Lucas-Hourani, M; Navratil, V; Meyniel, L; Achaz, G; Guironnet-Paquet, A; Aublin-Gex, A; Caignard, G; Cassonnet, P; Chaboud, A; Chantier, T; Deloire, A; Demeret, C; Le Breton, M; Neveu, G; Jacotot, L; Vaglio, P; Delmotte, S; Gautier, C; Combet, C; Deleage, G; Favre, M; Tangy, F; Jacob, Y; Andre, P; Lotteau, V; Rabourdin-Combe, C; Vidalain, P O

    2010-01-01

    Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

  4. Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

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    Fan Changyu

    2011-06-01

    Full Text Available Abstract Background Recent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the C. reinhardtii genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI, prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale. Results We assigned enzymatic functions to the translated JGI ORF models of C. reinhardtii by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST. In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate. Conclusions We functionally

  5. From the ORFeome concept to highly comprehensive, full-genome screening libraries.

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    Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

    2013-02-01

    Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries. PMID:22621725

  6. Archaeal extrachromosomal genetic elements

    DEFF Research Database (Denmark)

    Wang, Haina; Peng, Nan; Shah, Shiraz Ali;

    2015-01-01

    viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered...

  7. Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae

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    Maier Christina J

    2012-11-01

    Full Text Available Abstract Background The Gram-negative bacterium Chlamydia pneumoniae (Cpn is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. Results Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029 in Gateway® ‘entry’ vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway® entry clones (83% coverage and an additional 168 PCR-verified ‘pooled’ entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. Conclusion This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host–pathogen analysis. The high yield of

  8. Closing in on the C. elegans ORFeome by cloning TWINSCAN predictions

    DEFF Research Database (Denmark)

    Wei, Chaochun; Lamesch, Philippe; Arumugam, Manimozhiyan;

    2005-01-01

    The genome of Caenorhabditis elegans was the first animal genome to be sequenced. Although considerable effort has been devoted to annotating it, the standard WormBase annotation contains thousands of predicted genes for which there is no cDNA or EST evidence. We hypothesized that a more complete...... experimental annotation could be obtained by creating a more accurate gene-prediction program and then amplifying and sequencing predicted genes. Our approach was to adapt the TWINSCAN gene prediction system to C. elegans and C. briggsae and to improve its splice site and intron-length models. The resulting...... be significantly increased by replacing its partially curated predicted genes with TWINSCAN predictions. The technology described in this study will continue to drive the C. elegans ORFeome toward completion and contribute to the annotation of the three Caenorhabditis species currently being sequenced. The results...

  9. Archaeal DNA replication.

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    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  10. Archaeal viruses of the sulfolobales

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Garrett, Roger Antony

    2015-01-01

    Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environm......Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2...... in CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs...

  11. Shaping the Archaeal Cell Envelope

    NARCIS (Netherlands)

    Ellen, Albert F.; Zolghadr, Behnam; Driessen, Arnold M. J.; Albers, Sonja-Verena

    2010-01-01

    Although archaea have a similar cellular organization as other prokaryotes, the lipid composition of their membranes and their cell surface is unique. Here we discuss recent developments in our understanding of the archaeal protein secretion mechanisms, the assembly of macromolecular cell surface st

  12. Archaeal virus-host interactions

    NARCIS (Netherlands)

    Quax, T.E.F.

    2013-01-01

      The work presented in this thesis provides novel insights in several aspects of the molecular biology of archaea, bacteria and their viruses. Three fundamentally different groups of viruses are associated with the three domains of life. Archaeal viruses are characterized by a particularly

  13. Archaeal Enzymes and Applications in Industrial Biocatalysts.

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    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  14. Archaeal Enzymes and Applications in Industrial Biocatalysts

    Directory of Open Access Journals (Sweden)

    Jennifer A. Littlechild

    2015-01-01

    Full Text Available Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  15. The UCSC Archaeal Genome Browser: 2012 update

    OpenAIRE

    Chan, Patricia P.; Holmes, Andrew D.; Smith, Andrew M.; Tran, Danny; Lowe, Todd M.

    2011-01-01

    The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical web-based resource for exploration and discovery within archaeal and other selected microbial genomes. By bringing together existing gene annotations, gene expression data, multiple-genome alignments, pre-computed sequence comparisons and other specialized analysis tracks, the genome browser is a powerful aggregator of varied genomic information. The genome browser environment maintains the current look-and-feel of ...

  16. The UCSC Archaeal Genome Browser: 2012 update.

    Science.gov (United States)

    Chan, Patricia P; Holmes, Andrew D; Smith, Andrew M; Tran, Danny; Lowe, Todd M

    2012-01-01

    The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical web-based resource for exploration and discovery within archaeal and other selected microbial genomes. By bringing together existing gene annotations, gene expression data, multiple-genome alignments, pre-computed sequence comparisons and other specialized analysis tracks, the genome browser is a powerful aggregator of varied genomic information. The genome browser environment maintains the current look-and-feel of the vertebrate UCSC Genome Browser, but also integrates archaeal and bacterial-specific tracks with a few graphic display enhancements. The browser currently contains 115 archaeal genomes, plus 31 genomes of viruses known to infect archaea. Some of the recently developed or enhanced tracks visualize data from published high-throughput RNA-sequencing studies, the NCBI Conserved Domain Database, sequences from pre-genome sequencing studies, predicted gene boundaries from three different protein gene prediction algorithms, tRNAscan-SE gene predictions with RNA secondary structures and CRISPR locus predictions. We have also developed a companion resource, the Archaeal COG Browser, to provide better search and display of arCOG gene function classifications, including their phylogenetic distribution among available archaeal genomes.

  17. Protein Adaptations in Archaeal Extremophiles

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    Christopher J. Reed

    2013-01-01

    Full Text Available Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

  18. Archaeal Nitrification in Hot Springs

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    Richter, A.; Daims, H.; Reigstad, L.; Wanek, W.; Wagner, M.; Schleper, C.

    2006-12-01

    Biological nitrification, i.e. the aerobic conversion of ammonia to nitrate via nitrite, is a major component of the global nitrogen cycle. Until recently, it was thought that the ability to aerobically oxidize ammonia was confined to bacteria of the phylum Proteobacteria. However, it has recently been shown that Archaea of the phylum Crenarchaeota are also capable of ammonia oxidation. As many Crenarchaeota are thermophilic or hyperthermophilic, and at least some of them are capable of ammonia oxidation we speculated on the existence of (hyper)thermophilic ammonia-oxidizing archaea (AOA). Using PCR primers specifically targeting the archaeal ammonia monooxygenase (amoA) gene, we were indeed able to confirm the presence of such organisms in several hot springs in Reykjadalur, Iceland. These hot springs exhibited temperatures well above 80 °C and pH values ranging from 2.0 to 4.5. To proof that nitrification actually took place under these extreme conditions, we measured gross nitrification rates by the isotope pool dilution method; we added 15N-labelled nitrate to the mud and followed the dilution of the label by nitrate production from ammonium either in situ (incubation in the hot spring) or under controlled conditions in the laboratory (at 80 °C). The nitrification rates in the hot springs ranged from 0.79 to 2.22 mg nitrate-N per L of mud and day. Controls, in which microorganisms were killed before the incubations, demonstrated that the nitrification was of biological origin. Addition of ammonium increased the gross nitrification rate approximately 3-fold, indicating that the nitrification was ammonium limited under the conditions used. Collectively, our study provides evidence that (1) AOA are present in hot springs and (2) that they are actively nitrifying. These findings have major implications for our understanding of nitrogen cycling of hot environments.

  19. Hyperthermophilic Archaeal Viruses as Novel Nanoplatforms

    DEFF Research Database (Denmark)

    Uldahl, Kristine Buch

    ; attachment, alignment, and fusion. Upon infection, the intracellular replication cycle lasts 8 h at which point the virus particles are released as spindle-shaped tailless particles. Chapter II builds on the replication and purification methods in Chapter I to study the interaction between the two...... nanoplatforms than mammalian viruses because they cannot proliferate in humans and hence are less likely to trigger adverse effects. Another group of viruses that fits this criterion is archaeal viruses yet their potential remains untapped. As a group, archaeal viruses offer distinct advantages such as unique...

  20. Environmental shaping of sponge associated archaeal communities.

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    Aline S Turque

    Full Text Available BACKGROUND: Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum. CONCLUSION/SIGNIFICANCE: The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition

  1. Niche specialization of terrestrial archaeal ammonia oxidizers

    OpenAIRE

    Gubry-Rangin, Cécile; Hai, Brigitte; Quince, Christopher; Engel, Marion; Thomson, Bruce C.; James, Phillip; Schloter, Michael; Robert I. Griffiths; Prosser, James I.; Nicol, Graeme W.

    2011-01-01

    Soil pH is a major determinant of microbial ecosystem processes and potentially a major driver of evolution, adaptation, and diversity of ammonia oxidizers, which control soil nitrification. Archaea are major components of soil microbial communities and contribute significantly to ammonia oxidation in some soils. To determine whether pH drives evolutionary adaptation and community structure of soil archaeal ammonia oxidizers, sequences of amoA, a key functional gene of ammonia oxidation, were...

  2. TBP Domain Symmetry in Basal and Activated Archaeal Transcription

    OpenAIRE

    Ouhammouch, Mohamed; Hausner, Winfried; Geiduschek, E Peter

    2008-01-01

    The TATA-box binding protein (TBP) is the platform for assembly of archaeal and eukaryotic transcription preinitiation complexes. Ancestral gene duplication and fusion events have produced the saddle-shaped TBP molecule, with its two direct-repeat subdomains and pseudo-two-fold symmetry. Collectively, eukaryotic TBPs have diverged from their present-day archaeal counterparts, which remain highly symmetrical. The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in th...

  3. Sequence conserved for subcellular localization

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    Nair, Rajesh; Rost, Burkhard

    2002-01-01

    The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes. PMID:12441382

  4. Archaeal CRISPR-based immune systems

    DEFF Research Database (Denmark)

    Garrett, Roger A; Vestergaard, Gisle Alberg; Shah, Shiraz Ali

    2011-01-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-based immune systems are essentially modular with three primary functions: the excision and integration of new spacers, the processing of CRISPR transcripts to yield mature CRISPR RNAs (crRNAs), and the targeting and cleavage...... of foreign nucleic acid. The primary target appears to be the DNA of foreign genetic elements, but the CRISPR/Cmr system that is widespread amongst archaea also specifically targets and cleaves RNA in vitro. The archaeal CRISPR systems tend to be both diverse and complex. Here we examine evidence...... of CRISPR loci and the evidence for intergenomic exchange of CRISPR systems....

  5. Subcellular localization of pituitary enzymes

    Science.gov (United States)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  6. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  7. A Method for Identification of Selenoprotein Genes in Archaeal Genomes

    Institute of Scientific and Technical Information of China (English)

    Mingfeng Li; Yanzhao Huang; Yi Xiao

    2009-01-01

    The genetic codon UGA has a dual function: serving as a terminator and encoding selenocysteine. However, most popular gene annotation programs only take it as a stop signal, resulting in misannotation or completely missing selenoprotein genes. We developed a computational method named Asec-Prediction that is specific for the prediction of archaeal selenoprotein genes. To evaluate its effectiveness, we first applied it to 14 archaeal genomes with previously known selenoprotein genes, and Asec-Prediction identified all reported selenoprotein genes without redundant results. When we applied it to 12 archaeal genomes that had not been researched for selenoprotein genes, Asec-Prediction detected a novel selenoprotein gene in Methanosarcina acetivorans. Further evidence was also collected to support that the predicted gene should be a real selenoprotein gene. The result shows that Asec-Prediction is effective for the prediction of archaeal selenoprotein genes.

  8. Structure and Cell Biology of Archaeal Virus STIV

    OpenAIRE

    Fu, Chi-yu; Johnson, John E.

    2012-01-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interaction...

  9. Crystal structure of an archaeal actin homolog.

    Science.gov (United States)

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  10. Familial relationships in hyperthermo- and acidophilic archaeal viruses

    DEFF Research Database (Denmark)

    Happonen, Lotta Johanna; Redder, Peter; Peng, Xu;

    2010-01-01

    Archaea often live in extreme, harsh environments such as acidic hot springs and hypersaline waters. To date, only two icosahedrally symmetric, membrane-containing archaeal viruses, SH1 and Sulfolobus turreted icosahedral virus (STIV), have been described in detail. We report the sequence and three...

  11. Recent advances in imaging subcellular processes.

    Science.gov (United States)

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  12. Influences of plant type on bacterial and archaeal communities in constructed wetland treating polluted river water.

    Science.gov (United States)

    Long, Yan; Yi, Hao; Chen, Sili; Zhang, Zhengke; Cui, Kai; Bing, Yongxin; Zhuo, Qiongfang; Li, Bingxin; Xie, Shuguang; Guo, Qingwei

    2016-10-01

    Both bacteria and archaeal communities can play important roles in biogeochemical processes in constructed wetland (CW) system. However, the influence of plant type on microbial community in surface water CW remains unclear. The present study investigated bacterial and archaeal communities in five surface water CW systems with different plant species. The abundance, richness, and diversity of both bacterial and archaeal communities considerably differed in these five CW systems. Compared with the other three CW systems, the CW systems planted with Vetiveria zizanioides or Juncus effusus L. showed much higher bacterial abundance but lower archaeal abundance. Bacteria outnumbered archaea in each CW system. Moreover, the CW systems planted with V. zizanioides or J. effusus L. had relatively lower archaeal but higher bacterial richness and diversity. In each CW system, bacterial community displayed much higher richness and diversity than archaeal community. In addition, a remarkable difference of both bacterial and archaeal community structures was observed in the five studied CW systems. Proteobacteria was the most abundant bacterial group (accounting for 33-60 %). Thaumarchaeota organisms (57 %) predominated in archaeal communities in CW systems planted with V. zizanioides or J. effusus L., while Woesearchaeota (23 or 24 %) and Euryarchaeota (23 or 15 %) were the major archaeal groups in CW systems planted with Cyperus papyrus or Canna indica L. Archaeal community in CW planted with Typha orientalis Presl was mainly composed of unclassified archaea. Therefore, plant type exerted a considerable influence on microbial community in surface water CW system. PMID:27392623

  13. Distribution of Archaeal and Bacterial communities in a subtropical reservoir

    Directory of Open Access Journals (Sweden)

    Laís Américo Soares

    2015-12-01

    Full Text Available Abstract Aim: Microbial communities play a central role in environmental process such as organic matter mineralization and the nutrient cycling process in aquatic ecosystems. Despite their ecological importance, variability of the structure of archaeal and bacterial communities in freshwater remains understudied. Methods In the present study we investigated the richness and density of archaea and bacteria in the water column and sediments of the Itupararanga Reservoir. We also evaluated the relationship between the communities and the biotic and abiotic characteristics. Samples were taken at five depths in the water column next to the dam and three depths next to the reservoir entrance. Results PCR-DGGE evaluation of the archaeal and bacterial communities showed that both were present in the water column, even in oxygenated conditions. Conclusions The density of the bacteria (qPCR was greater than that of the archaea, a result of the higher metabolic plasticity of bacteria compared with archaea.

  14. Prediction of novel archaeal enzymes from sequence-derived features

    DEFF Research Database (Denmark)

    Jensen, Lars Juhl; Skovgaard, Marie; Brunak, Søren

    2002-01-01

    The completely sequenced archaeal genomes potentially encode, among their many functionally uncharacterized genes, novel enzymes of biotechnological interest. We have developed a prediction method for detection and classification of enzymes from sequence alone (available at http://www.cbs.dtu.dk/......The completely sequenced archaeal genomes potentially encode, among their many functionally uncharacterized genes, novel enzymes of biotechnological interest. We have developed a prediction method for detection and classification of enzymes from sequence alone (available at http......://www.cbs.dtu.dk/services/ArchaeaFun/). The method does not make use of sequence similarity; rather, it relies on predicted protein features like cotranslational and posttranslational modifications, secondary structure, and simple physical/chemical properties....

  15. Global analysis of viral infection in an archaeal model system

    Directory of Open Access Journals (Sweden)

    Walid S. Maaty

    2012-12-01

    Full Text Available The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes.. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled 6 times over a 72 hr period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change by nearly two-fold (p<0.05 with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 COG groups. 2D gel analysis showed that changes in post translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR associated proteins (CAS proteins were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP profiling on 2D gels showed caspase, hydrolase and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the

  16. Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil

    Directory of Open Access Journals (Sweden)

    Yendi E. Navarro-Noya

    2015-01-01

    Full Text Available In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5, indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena, and Natronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances clearly clustered the communities by pH.

  17. Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil.

    Science.gov (United States)

    Navarro-Noya, Yendi E; Valenzuela-Encinas, César; Sandoval-Yuriar, Alonso; Jiménez-Bueno, Norma G; Marsch, Rodolfo; Dendooven, Luc

    2015-01-01

    In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC) ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5), indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena, and Natronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances) clearly clustered the communities by pH.

  18. Ribonucleoproteins in Archaeal Pre-rRNA Processing and Modification

    Directory of Open Access Journals (Sweden)

    W. S. Vincent Yip

    2013-01-01

    Full Text Available Given that ribosomes are one of the most important cellular macromolecular machines, it is not surprising that there is intensive research in ribosome biogenesis. Ribosome biogenesis is a complex process. The maturation of ribosomal RNAs (rRNAs requires not only the precise cleaving and folding of the pre-rRNA but also extensive nucleotide modifications. At the heart of the processing and modifications of pre-rRNAs in Archaea and Eukarya are ribonucleoprotein (RNP machines. They are called small RNPs (sRNPs, in Archaea, and small nucleolar RNPs (snoRNPs, in Eukarya. Studies on ribosome biogenesis originally focused on eukaryotic systems. However, recent studies on archaeal sRNPs have provided important insights into the functions of these RNPs. This paper will introduce archaeal rRNA gene organization and pre-rRNA processing, with a particular focus on the discovery of the archaeal sRNP components, their functions in nucleotide modification, and their structures.

  19. Distribution and Diversity of Archaeal Ammonia Monooxygenase Genes Associated with Corals▿ †

    OpenAIRE

    Beman, J. Michael; Roberts, Kathryn J.; Wegley, Linda; Rohwer, Forest; Francis, Christopher A.

    2007-01-01

    Corals are known to harbor diverse microbial communities of Bacteria and Archaea, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A (amoA) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial amoA genes, and archaeal amoA gene sequences were obtained from five different species of coral coll...

  20. Bacterial and Archaeal Diversity From the Eastern Lau Spreading Center

    Science.gov (United States)

    Reysenbach, A.; Banta, A.; Kelly, S.; Kirshstein, J.; Voytek, M.

    2005-12-01

    Due to the diversity of venting styles, geological settings and variations in fluid geochemistry, the Valu Fa Ridge and Eastern Lau Spreading Center (ELSC) provide a unique opportunity to explore the effects geological and geochemical variables on patterns of microbial phylogenetic and metabolic diversity. High temperature sulfides, diffuse flow fluids and microbial mats were collected from six active vent fields on the Valu Fa Ridge and Eastern Lau Spreading Center during the R/V Melville cruise TUIM05MV. All samples were subsampled for molecular and microbial culturing purposes. The archaeal and bacterial 16S rRNA genes were amplified by PCR from a selection of samples. Additionally, the presence of Aquificales and an unidentified lineage, the DHVE archaeal group, was explored using PCR primers specific for these groups. A selection of DNAs were also screened for functional genes that are diagnostic for certain pathways, viz, aclB (reductive TCA cycle), mcrA (methanogenesis), nirS and nirK (nitrite reduction), amoA (ammonia oxidation). Culturing of thermophiles, both acidophiles and neutrophiles, was initiated. Over 20 hydrogen oxidizing (hydrogen and oxygen) or nitrate reducing (hydrogen and nitrate) chemolithoautotrophs were isolated as colonies and grow at 70 degrees C. All are related to Persephonella hydrogenophila, with the exception of 2 cultures that perhaps represent new species of Hydrogenivirga and Aquifex. Preliminary analysis of patterns of Aquificales diversity using both culturing and molecular approaches suggest that the distributions of this group alone are very different from that observed at other hydrothermal sites such as along the East Pacific Rise or Central Indian Ridge. As yet, the most commonly isolated Aquificales, P. marina, has not been detected in enrichment cultures from ELSC, and the diversity of Aquificales-related sequences is much greater than detected from sites along the EPR. It is therefore also likely, that patterns of

  1. Electroporation of archaeal lipid membranes using MD simulations.

    Science.gov (United States)

    Polak, Andraž; Tarek, Mounir; Tomšič, Matija; Valant, Janez; Ulrih, Nataša Poklar; Jamnik, Andrej; Kramar, Peter; Miklavčič, Damijan

    2014-12-01

    Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91 mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343 K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2 V to 4.3 V at 323 K, and to 5.2 V at 298 K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5 V at 323 K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50 mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8 V and 4.1 V at respectively 323 and 298 K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

  2. Factors affecting Archaeal Lipid Compositions of the Sulfolobus Species

    Science.gov (United States)

    He, L.; Han, J.; Wei, Y.; Lin, L.; Wei, Y.; Zhang, C.

    2010-12-01

    Temperature is the best known variable affecting the distribution of the archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in marine and freshwater systems. Other variables such as pH, ionic strength, or bicarbonate concentration may also affect archaeal GDGTs in terrestrial systems. Studies of pure cultures can help us pinpoint the specific effects these variables may have on archaeal lipid distribution in natural environments. In this study, three Sulfolobus species (HG4, HB5-2, HB9-6) isolated from Tengchong hot springs (pH 2-3, temperature 73-90°C) in China were used to investigate the effects of temperature, pH, substrate, and type of strain on the composition of GDGTs. Results showed that increase in temperature had negative effects on the relative contents of GDGT-0 (no cyclopentyl rings), GDGT-1 (one cyclopentyl ring), GDGT-2 and GDGT-3 but positive effects on GDGT-4, GDGT-4', GDGT-5 and GDGT-5'. Increase in pH, on the other hand, had negative effects on GDGT-0, GDGT-1, GDGT-4', GDGT-5 and GDGT-5', and positive effects on GDGT-3 and GDGT-4. GDGT-2 remained relatively constant with changing pH. When the HG4 was grown on different substrates, GDGT-5 was five time more abundant in sucrose-grown cultures than in yeast extract- or sulfur- grown cultures, suggesting that carbohydrates may stimulate the production of GDGT-5. For all three species, the ring index (average number of rings) of GDGTs correlated positively with incubation temperature. In HG4, ring index was much lower at optimal pH (3.5) than at other pH values. Ring index of HB5-2 or HB9-6 is higher than that of HG4, suggesting that speciation may affect the degree of cyclization of GDGT of the Sulfolobus. These results indicate that individual archaeal lipids respond differently to changes in environmental variables, which may be also species specific.

  3. Modelling the evolution of the archaeal tryptophan synthase

    Directory of Open Access Journals (Sweden)

    Merkl Rainer

    2007-04-01

    Full Text Available Abstract Background Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2 occur in different combinations. The evolutionary history of these trpB genes is under debate. Results In order to study the evolution of trp genes, completely sequenced archaeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operon-based trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i consisted of trpB2 genes located inside, the other (trpB2_a of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archaeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium

  4. The archaeal TFIIE homologue facilitates transcription initiation by enhancing TATA-box recognition

    NARCIS (Netherlands)

    Bell, S.D.; Brinkman, A.B.; Oost, van der J.; Jackson, S.P.

    2001-01-01

    Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)—homologues of eukaryal TBP and TFIIB—together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the pre

  5. Energy for two: New archaeal lineages and the origin of mitochondria.

    Science.gov (United States)

    Martin, William F; Neukirchen, Sinje; Zimorski, Verena; Gould, Sven B; Sousa, Filipa L

    2016-09-01

    Metagenomics bears upon all aspects of microbiology, including our understanding of mitochondrial and eukaryote origin. Recently, ribosomal protein phylogenies show the eukaryote host lineage - the archaeal lineage that acquired the mitochondrion - to branch within the archaea. Metagenomic studies are now uncovering new archaeal lineages that branch more closely to the host than any cultivated archaea do. But how do they grow? Carbon and energy metabolism as pieced together from metagenome assemblies of these new archaeal lineages, such as the Deep Sea Archaeal Group (including Lokiarchaeota) and Bathyarchaeota, do not match the physiology of any cultivated microbes. Understanding how these new lineages live in their environment is important, and might hold clues about how mitochondria arose and how the eukaryotic lineage got started. Here we look at these exciting new metagenomic studies, what they say about archaeal physiology in modern environments, how they impact views on host-mitochondrion physiological interactions at eukaryote origin. PMID:27339178

  6. Analyses of in vivo interactions between transcription factors and the archaeal RNA polymerase.

    Science.gov (United States)

    Walker, Julie E; Santangelo, Thomas J

    2015-09-15

    Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation.

  7. Methanobacterium Dominates Biocathodic Archaeal Communities in Methanogenic Microbial Electrolysis Cells

    KAUST Repository

    Siegert, Michael

    2015-07-06

    © 2015 American Chemical Society. Methane is the primary end product from cathodic current in microbial electrolysis cells (MECs) in the absence of methanogenic inhibitors, but little is known about the archaeal communities that develop in these systems. MECs containing cathodes made from different materials (carbon brushes, or plain graphite blocks or blocks coated with carbon black and platinum, stainless steel, nickel, ferrihydrite, magnetite, iron sulfide, or molybdenum disulfide) were inoculated with anaerobic digester sludge and acclimated at a set potential of -600 mV (versus a standard hydrogen electrode). The archaeal communities on all cathodes, except those coated with platinum, were predominated by Methanobacterium (median 97% of archaea). Cathodes with platinum contained mainly archaea most similar to Methanobrevibacter. Neither of these methanogens were abundant (<0.1% of archaea) in the inoculum, and therefore their high abundance on the cathode resulted from selective enrichment. In contrast, bacterial communities on the cathode were more diverse, containing primarily δ-Proteobacteria (41% of bacteria). The lack of a consistent bacterial genus on the cathodes indicated that there was no similarly selective enrichment of bacteria on the cathode. These results suggest that the genus Methanobacterium was primarily responsible for methane production in MECs when cathodes lack efficient catalysts for hydrogen gas evolution. (Figure Presented).

  8. Structure and cell biology of archaeal virus STIV.

    Science.gov (United States)

    Fu, Chi-yu; Johnson, Johnson E

    2012-04-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication. PMID:22482708

  9. DBMLoc: a Database of proteins with multiple subcellular localizations

    Directory of Open Access Journals (Sweden)

    Zhou Yun

    2008-02-01

    Full Text Available Abstract Background Subcellular localization information is one of the key features to protein function research. Locating to a specific subcellular compartment is essential for a protein to function efficiently. Proteins which have multiple localizations will provide more clues. This kind of proteins may take a high proportion, even more than 35%. Description We have developed a database of proteins with multiple subcellular localizations, designated DBMLoc. The initial release contains 10470 multiple subcellular localization-annotated entries. Annotations are collected from primary protein databases, specific subcellular localization databases and literature texts. All the protein entries are cross-referenced to GO annotations and SwissProt. Protein-protein interactions are also annotated. They are classified into 12 large subcellular localization categories based on GO hierarchical architecture and original annotations. Download, search and sequence BLAST tools are also available on the website. Conclusion DBMLoc is a protein database which collects proteins with more than one subcellular localization annotation. It is freely accessed at http://www.bioinfo.tsinghua.edu.cn/DBMLoc/index.htm.

  10. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  11. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    Science.gov (United States)

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  12. A formal ontology of subcellular neuroanatomy

    Directory of Open Access Journals (Sweden)

    Stephen D Larson

    2007-11-01

    Full Text Available The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO, covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described.

  13. RNA-Based Assessment of Diversity and Composition of Active Archaeal Communities in the German Bight

    Directory of Open Access Journals (Sweden)

    Bernd Wemheuer

    2012-01-01

    Full Text Available Archaea play an important role in various biogeochemical cycles. They are known extremophiles inhabiting environments such as thermal springs or hydrothermal vents. Recent studies have revealed a significant abundance of Archaea in moderate environments, for example, temperate sea water. Nevertheless, the composition and ecosystem function of these marine archaeal communities is largely unknown. To assess diversity and composition of active archaeal communities in the German Bight, seven marine water samples were taken and studied by RNA-based analysis of ribosomal 16S rRNA. For this purpose, total RNA was extracted from the samples and converted to cDNA. Archaeal community structures were investigated by pyrosequencing-based analysis of 16S rRNA amplicons generated from cDNA. To our knowledge, this is the first study combining next-generation sequencing and metatranscriptomics to study archaeal communities in marine habitats. The pyrosequencing-derived dataset comprised 62,045 archaeal 16S rRNA sequences. We identified Halobacteria as the predominant archaeal group across all samples with increased abundance in algal blooms. Thermoplasmatales (Euryarchaeota and the Marine Group I (Thaumarchaeota were identified in minor abundances. It is indicated that archaeal community patterns were influenced by environmental conditions.

  14. Magnetic Au Nanoparticles on Archaeal S-Layer Ghosts as Templates

    Directory of Open Access Journals (Sweden)

    Sonja Selenska-Pobell

    2011-10-01

    Full Text Available Cell‐ghosts representing empty cells of the archaeon Sulfolobus acidocaldarius, consisting only of their highly ordered and unusually stable outermost proteinaceous surface layer (S‐layer, were used as templates for Au nanoparticles fabrication. The properties of these archaeal Au nanoparticles differ significantly from those produced earlier by us onto bacterial S‐layer sheets. The archaeal Au nanoparticles, with a size of about 2.5 nm, consist exclusively of metallic Au(0, while those produced on the bacterial S‐layer had a size of about 4 nm and represented a mixture of Au(0 and Au(III in the ratio of 40 to 60 %. The most impressive feature of the archaeal Au nanoparticles is that they are strongly paramagnetic, in contrast to the bacterial ones and also to bulk gold. SQUID magnetometry and XMCD measurements demonstrated that the archaeal Au nanoparticles possess a rather large magnetic moment of about 0.1 µB/atom. HR‐ TEM‐EDX analysis revealed that the archaeal Au nanoparticles are linked to the sulfur atoms of the thiol groups of the amino acid cysteine, characteristic only for archaeal S‐layers. This is the first study demonstrating the formation of such unusually strong magnetic Au nanoparticles on a non‐modified archaeal S‐layer.

  15. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    Science.gov (United States)

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  16. Object Type Recognition for Automated Analysis of Protein Subcellular Location

    OpenAIRE

    Zhao, Ting; Velliste, Meel; Boland, Michael V.; Murphy, Robert F.

    2005-01-01

    The new field of location proteomics seeks to provide a comprehensive, objective characterization of the subcellular locations of all proteins expressed in a given cell type. Previous work has demonstrated that automated classifiers can recognize the patterns of all major subcellular organelles and structures in fluorescence microscope images with high accuracy. However, since some proteins may be present in more than one organelle, this paper addresses a more difficult task: recognizing a pa...

  17. Automated analysis of protein subcellular location in time series images

    OpenAIRE

    Hu, Yanhua; Osuna-Highley, Elvira; Hua, Juchang; Nowicki, Theodore Scott; Stolz, Robert; McKayle, Camille; Murphy, Robert F.

    2010-01-01

    Motivation: Image analysis, machine learning and statistical modeling have become well established for the automatic recognition and comparison of the subcellular locations of proteins in microscope images. By using a comprehensive set of features describing static images, major subcellular patterns can be distinguished with near perfect accuracy. We now extend this work to time series images, which contain both spatial and temporal information. The goal is to use temporal features to improve...

  18. Subcellular controls of mercury trophic transfer to a marine fish

    Energy Technology Data Exchange (ETDEWEB)

    Dang Fei [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2010-09-15

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  19. Subcellular controls of mercury trophic transfer to a marine fish

    International Nuclear Information System (INIS)

    Different behaviors of inorganic mercury [Hg(II)] and methylmercury (MeHg) during trophic transfer along the marine food chain have been widely reported, but the mechanisms are not fully understood. The bioavailability of ingested mercury, quantified by assimilation efficiency (AE), was investigated in a marine fish, the grunt Terapon jarbua, based on mercury subcellular partitioning in prey and purified subcellular fractions of prey tissues. The subcellular distribution of Hg(II) differed substantially among prey types, with cellular debris being a major (49-57% in bivalves) or secondary (14-19% in other prey) binding pool. However, MeHg distribution varied little among prey types, with most MeHg (43-79%) in heat-stable protein (HSP) fraction. The greater AEs measured for MeHg (90-94%) than for Hg(II) (23-43%) confirmed the findings of previous studies. Bioavailability of each purified subcellular fraction rather than the proposed trophically available metal (TAM) fraction could better elucidate mercury assimilation difference. Hg(II) associated with insoluble fraction (e.g. cellular debris) was less bioavailable than that in soluble fraction (e.g. HSP). However, subcellular distribution was shown to be less important for MeHg, with each fraction having comparable MeHg bioavailability. Subcellular distribution in prey should be an important consideration in mercury trophic transfer studies.

  20. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang;

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory...... element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...... mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression....

  1. Archaeal promoter architecture and mechanism of gene activation.

    Science.gov (United States)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang; She, Qunxin

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression. PMID:21265754

  2. Useful scars: Physics of the capsids of archaeal viruses

    Science.gov (United States)

    Perotti, L. E.; Dharmavaram, S.; Klug, W. S.; Marian, J.; Rudnick, J.; Bruinsma, R. F.

    2016-07-01

    We propose a physical model for the capsids of tailed archaeal viruses as viscoelastic membranes under tension. The fluidity is generated by thermal motion of scarlike structures that are an intrinsic feature of the ground state of large particle arrays covering surfaces with nonzero Gauss curvature. The tension is generated by a combination of the osmotic pressure of the enclosed genome and an extension force generated by filamentous structure formation that drives the formation of the tails. In continuum theory, the capsid has the shape of a surface of constant mean curvature: an unduloid. Particle arrays covering unduloids are shown to exhibit pronounced subdiffusive and diffusive single-particle transport at temperatures that are well below the melting temperature of defect-free particle arrays on a surface with zero Gauss curvature.

  3. Characterization of Olkiluoto bacterial and archaeal communities by 454 pyrosequencing

    Energy Technology Data Exchange (ETDEWEB)

    Bomberg, M.; Nyyssoenen, M.; Itaevaara, M. [VTT Technical Research Centre of Finland, Espoo (Finland)

    2012-06-15

    Recent advancement in sequencing technologies, 'Next Generation Sequencing', such as FLX 454 pyrosequencing has made it possible to obtain large amounts of sequence data where previously only few sequences could be obtained. This technique is especially useful for the study of community composition of uncultured microbial populations in environmental samples. In this project, the FLX 454 pyrosequencing technique was used to obtain up to 20 000 16S rRNA sequences or 10 000 mRNA sequences from each sample for identification of the microbial species composition as well as for comparison of the microbial communities between different samples. This project focused on the characterization of active microbial communities in the groundwater at the final disposal site of high radioactive wastes in Olkiluoto by FLX 454 pyrosequencing of the bacterial and archaeal ribosomal RNA as well as of the mRNA transcripts of the dsrB gene and mcrA gene of sulphate reducing bacteria and methanogenic archaea, respectively. Specific emphasis was put on studying the relationship of active and latent sulphate reducers and methanogens by qPCR due to their important roles in deep geobiochemical processes connected to copper corrosion. Seven packered boreholes were sampled anaerobically in Olkiluoto during 2009-2010. Groundwater was pumped from specific depths and the microbial cells werecollected by filtration on a membrane. Active microbial communities were studied based on RNA extracted from the membranes and translated to copy DNA, followed by sequencing by 454 Tag pyrosequencing. A total of 27 different bacterial and 17 archaeal taxonomic groups were detected.

  4. The Role of Multiple Transcription Factors In Archaeal Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Charles J. Daniels

    2008-09-23

    Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcanii was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of

  5. Geochemical Approach to Archaeal Ecology: δ13C of GDGTs

    Science.gov (United States)

    Lichtin, S.; Warren, C.; Pearson, A.; Pagani, M.

    2015-12-01

    Over the last decade and a half, glycerol dialkyl glycerol tetraethers (GDGTs) have increasingly been used to reconstruct environmental temperatures; proxies like TEX86 that correlate the relative abundance of these archaeal cell membrane lipids to sea surface temperature are omnipresent in paleoclimatology literature. While it has become common to make claims about past temperatures using GDGTs, our present understanding of the organisms that synthesize the compounds is still quite limited. The generally accepted theory states that microorganisms like the Thaumarchaeota modify the structure of membrane lipids to increase intermolecular interactions, strengthening the membrane at higher temperatures. Yet to date, culture experiments have been largely restricted to a single species, Nitrosopumilus maritimes, and recent studies on oceanic archaeal rRNA have revealed that these biomarkers are produced in diverse, heterogeneous, and site-specific communities. This brings up questions as to whether different subclasses of GDGTs, and all subsequent proxies, represent adaptation within a single organismal group or a shift in community composition. To investigate whether GDGTs with different chain structures, from the simple isoprenoidal GDGT-0 to Crenarchaeol with its many cyclopentane groups, are sourced from archaea with similar or disparate metabolic pathways—and if that information is inherited in GDGTs trapped in marine sediments—this study examines the stable carbon isotope values (δ13C) of GDGTs extracted from the uppermost meters of sediment in the Orca Basin, Gulf of Mexico, using spooling-wire microcombustion isotope-ratio mass spectrometer (SWiM-IRMS), tackling a fundamental assumption of the TEX86 proxy that influences the way we perceive the veracity of existing temperature records.

  6. Changes in northern Gulf of Mexico sediment bacterial and archaeal communities exposed to hypoxia

    Science.gov (United States)

    Biogeochemical changes in marine sediments during coastal water hypoxia are well described, but less is known about underlying changes in microbial communities. Bacterial and archaeal communities in Louisiana continental shelf (LCS) hypoxic zone sediments were characterized by py...

  7. The essence of being extremophilic : the role of the unique archaeal membrane lipids

    NARCIS (Netherlands)

    Vossenberg, Jack L.C.M. van de; Driessen, Arnold J.M.; Konings, Wil N.

    1998-01-01

    In extreme environments, mainly Archaea are encountered. The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant. Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spann

  8. Response of Archaeal Communities in Beach Sediments to Spilled Oil and Bioremediation

    OpenAIRE

    Röling, Wilfred F. M.; Couto de Brito, Ivana R.; Swannell, Richard P. J.; Head, Ian M.

    2004-01-01

    While the contribution of Bacteria to bioremediation of oil-contaminated shorelines is well established, the response of Archaea to spilled oil and bioremediation treatments is unknown. The relationship between archaeal community structure and oil spill bioremediation was examined in laboratory microcosms and in a bioremediation field trial. 16S rRNA gene-based PCR and denaturing gradient gel analysis revealed that the archaeal community in oil-free laboratory microcosms was stable for 26 day...

  9. Subcellular Proteomics of Soybean under Flooding Stress

    Institute of Scientific and Technical Information of China (English)

    Setsuko Komatsu

    2012-01-01

    Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate,and causes significant reductions in the growth and yield of several crops.The application of proteomics techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops.To understand the response mechanism of soybean under flooding stress,proteomics analysis was carried out.Especially,subcellular proteomics studies have led to a better understanding of the mechanism of flooding stress tolerance in soybean.The effects of flooding stress on root plasma membrane were analyzed using an aqueous two-phase partitioning method in combination with gel-based and gel-free proteomics techniques.The results led to the following conclusions:proteins located in the cell wall were increased in the plasma membrane of flooded plants,indicating the contribution of plasma membrane to modification of the cell wall; superoxide dismutase was increased,indicating that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to flooding stress; heat shock cognate 70 kDa protein likely plays a significant role in protecting other proteins from denaturation and degradation during flooding stress; and signaling proteins might work cooperatively to regulate plasma membrane H +-ATPase and maintain ion homeostasis.Cell wall proteins were isolated from root of flooding stressed plants via sucrose gradient centrifugation and analyzed using gel-based proteomics technique.Cell wall proteins identified were related to lignification,and these results indicated that a decrease of lignification-related proteins is related to flooding decreased ROS and jasmonate biosynthesis.And also,lignin staining confirmed that lignification was suppressed in the roots of flooding stressed soybeans.Mitochondrial fractions were purified from root of flooding stressed

  10. Predicting Protein Subcellular Localization: Past, Present, and Future

    Institute of Scientific and Technical Information of China (English)

    Pierre D(o)nnes; Annette H(o)glund

    2004-01-01

    Functional characterization of every single protein is a major challenge of the postgenomic era. The large-scale analysis of a cell's proteins, proteomics, seeks to provide these proteins with reliable annotations regarding their interaction partners and functions in the cellular machinery. An important step on this way is to determine the subcellular localization of each protein. Eukaryotic cells are divided into subcellular compartments, or organelles. Transport across the membrane into the organelles is a highly regulated and complex cellular process. Predicting the subcellular localization by computational means has been an area of vivid activity during recent years. The publicly available prediction methods differ mainly in four aspects: the underlying biological motivation, the computational method used, localization coverage, and reliability, which are of importance to the user.This review provides a short description of the main events in the protein sorting process and an overview of the most commonly used methods in this field.

  11. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  12. HECTAR: A method to predict subcellular targeting in heterokonts

    Directory of Open Access Journals (Sweden)

    Guermeur Yann

    2008-09-01

    Full Text Available Abstract Background The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem. Results HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine. Conclusion The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal

  13. Identification of an archaeal mercury regulon by chromatin immunoprecipitation.

    Science.gov (United States)

    Rudrappa, Deepak; Yao, Andrew I; White, Derrick; Pavlik, Benjamin J; Singh, Raghuveer; Facciotti, Marc T; Blum, Paul

    2015-12-01

    Mercury is a heavy metal and toxic to all forms of life. Metal exposure can invoke a response to improve survival. In archaea, several components of a mercury response system have been identified, but it is not known whether metal transport is a member of this system. To identify such missing components, a peptide-tagged MerR transcription factor was used to localize enriched chromosome regions by chromosome immunoprecipitation combined with DNA sequence analysis. Such regions could serve as secondary regulatory binding sites to control the expression of additional genes associated with mercury detoxification. Among the 31 highly enriched loci, a subset of five was pursued as potential candidates based on their current annotations. Quantitative reverse transcription-PCR analysis of these regions with and without mercury treatment in WT and mutant strains lacking merR indicated significant regulatory responses under these conditions. Of these, a Family 5 extracellular solute-binding protein and the MarR transcription factor shown previously to control responses to oxidation were most strongly affected. Inactivation of the solute-binding protein by gene disruption increased the resistance of mutant cells to mercury challenge. Inductively coupled plasma-MS analysis of the mutant cell line following metal challenge indicated there was less intracellular mercury compared with the isogenic WT strain. Together, these regulated genes comprise new members of the archaeal MerR regulon and reveal a cascade of transcriptional control not previously demonstrated in this model organism.

  14. Protein phosphorylation and its role in archaeal signal transduction.

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C; Albers, Sonja-Verena; Siebers, Bettina

    2016-09-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies.

  15. Protein phosphorylation and its role in archaeal signal transduction

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  16. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2016-01-01

    When predicting the subcellular localization of proteins from their amino acid sequences, there are basically three approaches: signal-based, global property-based, and homology-based. Each of these has its advantages and drawbacks, and it is important when comparing methods to know which approac...

  17. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement.

    Science.gov (United States)

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  18. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    Science.gov (United States)

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-01-01

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics. PMID:27078038

  19. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    Science.gov (United States)

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  20. Abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China.

    Science.gov (United States)

    Song, Zhao-Qi; Wang, Li; Wang, Feng-Ping; Jiang, Hong-Chen; Chen, Jin-Quan; Zhou, En-Min; Liang, Feng; Xiao, Xiang; Li, Wen-Jun

    2013-09-01

    It has been suggested that archaea carrying the accA gene, encoding the alpha subunit of the acetyl CoA carboxylase, autotrophically fix CO2 using the 3-hydroxypropionate/4-hydroxybutyrate pathway in low-temperature environments (e.g., soils, oceans). However, little new information has come to light regarding the occurrence of archaeal accA genes in high-temperature ecosystems. In this study, we investigated the abundance and diversity of archaeal accA gene in hot springs in Yunnan Province, China, using DNA- and RNA-based phylogenetic analyses and quantitative polymerase chain reaction. The results showed that archaeal accA genes were present and expressed in the investigated Yunnan hot springs with a wide range of temperatures (66-96 °C) and pH (4.3-9.0). The majority of the amplified archaeal accA gene sequences were affiliated with the ThAOA/HWCG III [thermophilic ammonia-oxidizing archaea (AOA)/hot water crenarchaeotic group III]. The archaeal accA gene abundance was very close to that of AOA amoA gene, encoding the alpha subunit of ammonia monooxygenase. These data suggest that AOA in terrestrial hot springs might acquire energy from ammonia oxidation coupled with CO2 fixation using the 3-hydroxypropionate/4-hydroxybutyrate pathway.

  1. Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways.

    Science.gov (United States)

    Gietl, Andreas; Holzmeister, Phil; Blombach, Fabian; Schulz, Sarah; von Voithenberg, Lena Voith; Lamb, Don C; Werner, Finn; Tinnefeld, Philip; Grohmann, Dina

    2014-06-01

    During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex. PMID:24744242

  2. pH dominates variation in tropical soil archaeal diversity and community structure.

    Science.gov (United States)

    Tripathi, Binu M; Kim, Mincheol; Lai-Hoe, Ang; Shukor, Nor A A; Rahim, Raha A; Go, Rusea; Adams, Jonathan M

    2013-11-01

    Little is known of the factors influencing soil archaeal community diversity and composition in the tropics. We sampled soils across a range of forest and nonforest environments in the equatorial tropics of Malaysia, covering a wide range of pH values. DNA was PCR-amplified for the V1-V3 region of the 16S rRNA gene, and 454-pyrosequenced. Soil pH was the best predictor of diversity and community composition of Archaea, being a stronger predictor than land use. Archaeal OTU richness was highest in the most acidic soils. Overall archaeal abundance in tropical soils (determined by qPCR) also decreased at higher pH. This contrasts with the opposite trend previously found in temperate soils. Thaumarcheota group 1.1b was more abundant in alkaline soils, whereas group 1.1c was only detected in acidic soils. These results parallel those found in previous studies in cooler climates, emphasizing niche conservatism among broad archaeal groups. Among the most abundant operational taxonomic units (OTUs), there was clear evidence of niche partitioning by pH. No individual OTU occurred across the entire range of pH values. Overall, the results of this study show that pH plays a major role in structuring tropical soil archaeal communities.

  3. Composition of bacterial and archaeal communities during landfill refuse decomposition processes.

    Science.gov (United States)

    Song, Liyan; Wang, Yangqing; Zhao, Heping; Long, David T

    2015-12-01

    Little is known about the archaeal and the bacterial diversities in a landfill during different phases of decomposition. In this study, the archaeal and the bacterial diversities of Laogang landfill (Shanghai, China) at two different decomposition phases (i.e., initial methanogenic phase (IMP) and stable methanogenic phase (SMP)), were culture-independently examined using PCR-based 454 pyrosequencing. A total of 47,753 sequences of 16S rRNA genes were retrieved from 69,954 reads and analyzed to evaluate the diversities of the archaeal and bacterial communities. The most predominant types of archaea were hydrogenotrophic Methanomicrobiales, and of bacteria were Proteobacteria, Firmicutes, and Bacteroidetes. As might be expected, their abundances varied at decomposition phases. Archaea Methanomicrobiales accounts for 97.6% of total archaeal population abundance in IMP and about 57.6% in SMP. The abundance of archaeal genus Halobacteriale was 0.1% in IMP and was 20.3% in the SMP. The abundance of Firmicutes was 21.3% in IMP and was 4.3% in SMP. The abundance of Bacteroidetes represented 11.5% of total bacterial in IMP and was dominant (49.4%) in SMP. Both the IMP and SMP had unique cellulolytic bacteria compositions. IMP consisted of members of Bacillus, Fibrobacter, and Eubacterium, while SMP harbored groups of Microbacterium. Both phases had Clostridium with different abundance, 4-5 folds higher in SMP.

  4. Virus-mediated archaeal hecatomb in the deep seafloor

    Science.gov (United States)

    Danovaro, Roberto; Dell’Anno, Antonio; Corinaldesi, Cinzia; Rastelli, Eugenio; Cavicchioli, Ricardo; Krupovic, Mart; Noble, Rachel T.; Nunoura, Takuro; Prangishvili, David

    2016-01-01

    Viruses are the most abundant biological entities in the world’s oceans, and they play a crucial role in global biogeochemical cycles. In deep-sea ecosystems, archaea and bacteria drive major nutrient cycles, and viruses are largely responsible for their mortality, thereby exerting important controls on microbial dynamics. However, the relative impact of viruses on archaea compared to bacteria is unknown, limiting our understanding of the factors controlling the functioning of marine systems at a global scale. We evaluate the selectivity of viral infections by using several independent approaches, including an innovative molecular method based on the quantification of archaeal versus bacterial genes released by viral lysis. We provide evidence that, in all oceanic surface sediments (from 1000- to 10,000-m water depth), the impact of viral infection is higher on archaea than on bacteria. We also found that, within deep-sea benthic archaea, the impact of viruses was mainly directed at members of specific clades of Marine Group I Thaumarchaeota. Although archaea represent, on average, ~12% of the total cell abundance in the top 50 cm of sediment, virus-induced lysis of archaea accounts for up to one-third of the total microbial biomass killed, resulting in the release of ~0.3 to 0.5 gigatons of carbon per year globally. Our results indicate that viral infection represents a key mechanism controlling the turnover of archaea in surface deep-sea sediments. We conclude that interactions between archaea and their viruses might play a profound, previously underestimated role in the functioning of deep-sea ecosystems and in global biogeochemical cycles. PMID:27757416

  5. Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

    NARCIS (Netherlands)

    Frade, P.R.; Roll, K.; Bergauer, K.; Herndl, G.

    2016-01-01

    Comparative studies on the distribution of archaeal versus bacterial communities associatedwith the surface mucus layer of corals have rarely taken place. It has thereforeremained enigmatic whether mucus-associated archaeal and bacterial communities exhibita similar specificity towards coral hosts a

  6. Liquid but Durable: Molecular Dynamics Simulations Explain the Unique Properties of Archaeal-Like Membranes

    Science.gov (United States)

    Chugunov, Anton O.; Volynsky, Pavel E.; Krylov, Nikolay A.; Boldyrev, Ivan A.; Efremov, Roman G.

    2014-12-01

    Archaeal plasma membranes appear to be extremely durable and almost impermeable to water and ions, in contrast to the membranes of Bacteria and Eucaryota. Additionally, they remain liquid within a temperature range of 0-100°C. These are the properties that have most likely determined the evolutionary fate of Archaea, and it may be possible for bionanotechnology to adopt these from nature. In this work, we use molecular dynamics simulations to assess at the atomistic level the structure and dynamics of a series of model archaeal membranes with lipids that have tetraether chemical nature and ``branched'' hydrophobic tails. We conclude that the branched structure defines dense packing and low water permeability of archaeal-like membranes, while at the same time ensuring a liquid-crystalline state, which is vital for living cells. This makes tetraether lipid systems promising in bionanotechnology and material science, namely for design of new and unique membrane nanosystems.

  7. Turnover of microbial lipids in the deep biosphere and growth of benthic archaeal populations.

    Science.gov (United States)

    Xie, Sitan; Lipp, Julius S; Wegener, Gunter; Ferdelman, Timothy G; Hinrichs, Kai-Uwe

    2013-04-01

    Deep subseafloor sediments host a microbial biosphere with unknown impact on global biogeochemical cycles. This study tests previous evidence based on microbial intact polar lipids (IPLs) as proxies of live biomass, suggesting that Archaea dominate the marine sedimentary biosphere. We devised a sensitive radiotracer assay to measure the decay rate of ([(14)C]glucosyl)-diphytanylglyceroldiether (GlcDGD) as an analog of archaeal IPLs in continental margin sediments. The degradation kinetics were incorporated in model simulations that constrained the fossil fraction of subseafloor IPLs and rates of archaeal turnover. Simulating the top 1 km in a generic continental margin sediment column, we estimated degradation rate constants of GlcDGD being one to two orders of magnitude lower than those of bacterial IPLs, with half-lives of GlcDGD increasing with depth to 310 ky. Given estimated microbial community turnover times of 1.6-73 ky in sediments deeper than 1 m, 50-96% of archaeal IPLs represent fossil signals. Consequently, previous lipid-based estimates of global subseafloor biomass probably are too high, and the widely observed dominance of archaeal IPLs does not rule out a deep biosphere dominated by Bacteria. Reverse modeling of existing concentration profiles suggest that archaeal IPL synthesis rates decline from around 1,000 pg⋅mL(-1) sediment⋅y(-1) at the surface to 0.2 pg⋅mL(-1)⋅y(-1) at 1 km depth, equivalent to production of 7 × 10(5) to 140 archaeal cells⋅mL(-1) sediment⋅y(-1), respectively. These constraints on microbial growth are an important step toward understanding the relationship between the deep biosphere and the carbon cycle.

  8. Differences in the Composition of Archaeal Communities in Sediments from Contrasting Zones of Lake Taihu

    Science.gov (United States)

    Fan, Xianfang; Xing, Peng

    2016-01-01

    In shallow lakes, different primary producers might impact the physiochemical characteristics of the sediment and the associated microbial communities. Until now, little was known about the features of sediment Archaea and their variation across different primary producer-dominated ecosystems. Lake Taihu provides a suitable study area with cyanobacteria- and macrophyte-dominated zones co-occurring in one ecosystem. The composition of the sediment archaeal community was assessed using 16S rRNA gene amplicon sequencing technology, based on which the potential variation with respect to the physiochemical characteristics of the sediment was analyzed. Euryarchaeota (30.19% of total archaeal sequences) and Bathyarchaeota (28.00%) were the two most abundant phyla, followed by Crenarchaeota (11.37%), Aigarchaeota (10.24%) and Thaumarchaeota (5.98%). The differences found in the composition of the archaeal communities between the two zones was significant (p = 0.005). Sediment from macrophyte-dominated zones had high TOC and TN content and an abundance of archaeal lineages potentially involved in the degradation of complex organic compounds, such as the order Thermoplasmatales. In the area dominated by Cyanobacteria, archaeal lineages related to sulfur metabolism, for example, Sulfolobales and Desulfurococcales, were significantly enriched. Among Bathyarchaeota, subgroups MCG-6 and MCG-15 were significantly accumulated in the sediment of areas dominated by macrophytes whereas MCG-4 was consistently dominant in both type of sediments. The present study contributes to the knowledge of sediment archaeal communities with different primary producers and their possible biogeochemical functions in sediment habitats. PMID:27708641

  9. Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring

    Directory of Open Access Journals (Sweden)

    Sonu Bhatia

    2015-09-01

    Full Text Available The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche.

  10. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    Science.gov (United States)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  11. Imaging trace element distributions in single organelles and subcellular features

    Science.gov (United States)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  12. Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

    CERN Document Server

    Zurbuchen, Mark A; Kohan, Sirus A; Leung, Belinda; Bouchard, Louis-S

    2015-01-01

    There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a subcellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable "zooming-in" to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of...

  13. Evaluation and comparison of mammalian subcellular localization prediction methods

    Directory of Open Access Journals (Sweden)

    Fink J Lynn

    2006-12-01

    Full Text Available Abstract Background Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER, peroxisome, and lysosome. The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE

  14. Subcellular site and nature of intracellular cadmium in plants

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, George J.

    1979-01-01

    The mechanisms underlying heavy metal accumulation, toxicity and tolerance in higher plants are poorly understood. Since subcellular processes are undoubtedly involved in all these phenomena, it is of interest to study the extent of, subcellular site of and nature of intracellularly accumulated cadmium in higher plants. Whole plants supplied /sup 109/CdCl/sub 2/ or /sup 112/CdSO/sub 4/ accumulated Cd into roots and aerial tissues. Preparation of protoplasts from aerial tissue followed by subcellular fractionation of the protoplasts to obtain intact vacuoles, chloroplasts and cytosol revealed the presence of Cd in the cytosol but not in vacuoles or chloroplasts. Particulate materials containing other cell components were also labeled. Of the /sup 109/Cd supplied to plants, 2 to 10% was recovered in both cytosol preparations and in particulate materials. Cytosol contained proteinaceous--Cd complexes, free metal and low molecular weight Cd complexes. Labeling of protoplasts gave similar results. No evidence was obtained for the production of volatile Cd complexes in tobacco.

  15. Planktonic Euryarchaeota are a significant source of archaeal tetraether lipids in the ocean

    Science.gov (United States)

    Lincoln, Sara A.; Wai, Brenner; Eppley, John M.; Church, Matthew J.; Summons, Roger E.; DeLong, Edward F.

    2014-01-01

    Archaea are ubiquitous in marine plankton, and fossil forms of archaeal tetraether membrane lipids in sedimentary rocks document their participation in marine biogeochemical cycles for >100 million years. Ribosomal RNA surveys have identified four major clades of planktonic archaea but, to date, tetraether lipids have been characterized in only one, the Marine Group I Thaumarchaeota. The membrane lipid composition of the other planktonic archaeal groups—all uncultured Euryarchaeota—is currently unknown. Using integrated nucleic acid and lipid analyses, we found that Marine Group II Euryarchaeota (MG-II) contributed significantly to the tetraether lipid pool in the North Pacific Subtropical Gyre at shallow to intermediate depths. Our data strongly suggested that MG-II also synthesize crenarchaeol, a tetraether lipid previously considered to be a unique biomarker for Thaumarchaeota. Metagenomic datasets spanning 5 y indicated that depth stratification of planktonic archaeal groups was a stable feature in the North Pacific Subtropical Gyre. The consistent prevalence of MG-II at depths where the bulk of exported organic matter originates, together with their ubiquitous distribution over diverse oceanic provinces, suggests that this clade is a significant source of tetraether lipids to marine sediments. Our results are relevant to archaeal lipid biomarker applications in the modern oceans and the interpretation of these compounds in the geologic record. PMID:24946804

  16. The effect of maturity and depositional redox conditions on archaeal tetraether lipid palaeothermometry

    NARCIS (Netherlands)

    Sinninghe Damsté, J.S.; Schouten, S.; Hopmans, E.C.

    2004-01-01

    Recently we proposed a new organic sea surface temperature proxy, TEX86, based on the distribution of archaeal tetraether lipids. Here, we have examined the effect of oxic degradation and maturity on this new temperature proxy. Our results show that oxic degradation does not appear to affect the TEX

  17. Effect of soil properties and hydrology on Archaeal community composition in three temperate grasslands on peat

    DEFF Research Database (Denmark)

    Görres, Carolyn-Monika; Conrad, Ralf; Petersen, Søren O

    2013-01-01

    Grasslands established on drained peat soils are regarded as negligible methane (CH4) sources; however, they can still exhibit considerable soil CH4 dynamics. We investigated archaeal community composition in two different fen peat soils and one bog peat soil under permanent grassland in Denmark...

  18. Archaeal and bacterial diversity in hot springs on the Tibetan Plateau, China.

    Science.gov (United States)

    Huang, Qiuyuan; Dong, Christina Z; Dong, Raymond M; Jiang, Hongchen; Wang, Shang; Wang, Genhou; Fang, Bin; Ding, Xiaoxue; Niu, Lu; Li, Xin; Zhang, Chuanlun; Dong, Hailiang

    2011-09-01

    The diversity of archaea and bacteria was investigated in ten hot springs (elevation >4600 m above sea level) in Central and Central-Eastern Tibet using 16S rRNA gene phylogenetic analysis. The temperature and pH of these hot springs were 26-81°C and close to neutral, respectively. A total of 959 (415 and 544 for bacteria and archaea, respectively) clone sequences were obtained. Phylogenetic analysis showed that bacteria were more diverse than archaea and that these clone sequences were classified into 82 bacterial and 41 archaeal operational taxonomic units (OTUs), respectively. The retrieved bacterial clones were mainly affiliated with four known groups (i.e., Firmicutes, Proteobacteria, Cyanobacteria, Chloroflexi), which were similar to those in other neutral-pH hot springs at low elevations. In contrast, most of the archaeal clones from the Tibetan hot springs were affiliated with Thaumarchaeota, a newly proposed archaeal phylum. The dominance of Thaumarchaeota in the archaeal community of the Tibetan hot springs appears to be unique, although the exact reasons are not yet known. Statistical analysis showed that diversity indices of both archaea and bacteria were not statistically correlated with temperature, which is consistent with previous studies.

  19. Metagenomic evaluation of bacterial and archaeal diversity in the geothermal hot springs of manikaran, India.

    Science.gov (United States)

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Green, Stefan J; Joshi, Amit; Chauhan, Ashvini

    2015-02-19

    Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat.

  20. CrAgDb--a database of annotated chaperone repertoire in archaeal genomes.

    Science.gov (United States)

    Rani, Shikha; Srivastava, Abhishikha; Kumar, Manish; Goel, Manisha

    2016-03-01

    Chaperones are a diverse class of ubiquitous proteins that assist other cellular proteins in folding correctly and maintaining their native structure. Many different chaperones cooperate to constitute the 'proteostasis' machinery in the cells. It has been proposed earlier that archaeal organisms could be ideal model systems for deciphering the basic functioning of the 'protein folding machinery' in higher eukaryotes. Several chaperone families have been characterized in archaea over the years but mostly one protein at a time, making it difficult to decipher the composition and mechanistics of the protein folding system as a whole. In order to deal with these lacunae, we have developed a database of all archaeal chaperone proteins, CrAgDb (Chaperone repertoire in Archaeal genomes). The data have been presented in a systematic way with intuitive browse and search facilities for easy retrieval of information. Access to these curated datasets should expedite large-scale analysis of archaeal chaperone networks and significantly advance our understanding of operation and regulation of the protein folding machinery in archaea. Researchers could then translate this knowledge to comprehend the more complex protein folding pathways in eukaryotic systems. The database is freely available at http://14.139.227.92/mkumar/cragdb/. PMID:26862144

  1. Identification of archaeal proteins that affect the exosome function in vitro

    Directory of Open Access Journals (Sweden)

    Palhano Fernando L

    2010-05-01

    Full Text Available Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

  2. Seasonal Effects in a Lake Sediment Archaeal Community of the Brazilian Savanna

    Directory of Open Access Journals (Sweden)

    Thiago Rodrigues

    2014-01-01

    Full Text Available The Cerrado is a biome that corresponds to 24% of Brazil’s territory. Only recently microbial communities of this biome have been investigated. Here we describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerrado in the dry season and in the transition period between the dry and rainy seasons, when the first rains occur. Gene libraries were constructed, using Archaea-specific primers for the 16S rRNA and amoA genes. Analysis revealed marked differences between the archaeal communities found in the two seasons. I.1a and I.1c Thaumarchaeota were found in greater numbers in the transition period, while MCG Archaea was dominant on the dry season. Methanogens were only found in the dry season. Analysis of 16S rRNA sequences revealed lower diversity on the transition period. We detected archaeal amoA sequences in both seasons, but there were more OTUs during the dry season. These sequences were within the same cluster as Nitrosotalea devanaterra’s amoA gene. The principal coordinate analysis (PCoA test revealed significant differences between samples from different seasons. These results provide information on archaeal diversity in freshwater lake sediments of the Cerrado and indicates that rain is likely a factor that impacts these communities.

  3. Archaeal communities associated with roots of the common reed (Phragmites australis) in Beijing Cuihu Wetland.

    Science.gov (United States)

    Liu, Yin; Li, Hong; Liu, Qun Fang; Li, Yan Hong

    2015-05-01

    The richness, phylogeny and composition of archaeal community associated with the roots of common reed (Phragmites australis) growing in the Beijing Cuihu Wetland, China was investigated using a 16S rDNA library. In total, 235 individual sequences were collected, and a phylogenetic analysis revealed that 69.4 and 11.5 % of clones were affiliated with the Euryarchaeota and the Crenarchaeota, respectively. In Euryarchaeota, the archaeal community was dominated by species in following genera: Methanobacterium in the order Methanobacteriales (60.7 %); Methanoregula and Methanospirillum in the order Methanomicrobiales (20.2 %), and Methanomethylovorans, Methanosarcina and Methanosaeta in the order Methanosarcinales (17.2 %). Of 27 sequences assigned to uncultured Crenarchaeota, 22 were grouped into Group 1.3, and five grouped into Group 1.1b. Hence, the archaeal communities associated with reed roots are largely involved in methane production, and, to a lesser extent, in ammonia oxidization. Quantification of the archaeal amoA gene indicated that ammonia oxidizing archaea were more numerous in the rhizosphere soil than in the root tissue or surrounding water. A total of 19.1 % of the sequences were unclassified, suggesting that many unidentified archaea are probably involved in the reed wetland ecosystem. PMID:25739566

  4. ENERGY-TRANSDUCING PROPERTIES OF PRIMARY PROTON PUMPS RECONSTITUTED INTO ARCHAEAL BIPOLAR LIPID VESICLES

    NARCIS (Netherlands)

    ELFERINK, MGL; DEWIT, JG; DRIESSEN, AJM; KONINGS, WN; Elferink, Marieke G.L.

    1993-01-01

    Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties. From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aq

  5. Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria

    Directory of Open Access Journals (Sweden)

    Galina Radeva

    2014-01-01

    Full Text Available Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA. Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

  6. Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards.

    Science.gov (United States)

    Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

    2014-11-01

    It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profiling. The abundances of total bacteria, total archaea, and selected functional genes (bacterial and archaeal amoA, bacterial narG, nirK, nirS, and nosZ) were determined by quantitative polymerase chain reaction (qPCR). The results indicate that the structures of bacterial and archaeal communities varied significantly with time and fertilization based on changes in the relative abundance of dominant T-RFs. The abundancy of the detected genes changed with time. The total bacteria, total archaea, and archaeal amoA were less abundant in July. The bacterial amoA and denitrifying genes were less abundant in September, except the nirK gene. The OF treatment increased the abundance of the observed genes, while the CF treatment had little influence on them. The soil temperature significantly affected the bacterial and archaeal community structures. The soil moisture was significantly correlated with the abundance of denitrifying genes. Of the soil chemical properties, soil organic carbon was the most important factor and was significantly correlated with the abundance of the detected genes, except the nirK gene. Overall, this study demonstrated the effects of both temporal alteration and organic fertilizer on the structures of microbial communities and the abundance of genes involved in the nitrogen cycle.

  7. Structure of the rare archaeal biosphere and seasonal dynamics of active ecotypes in surface coastal waters.

    Science.gov (United States)

    Hugoni, Mylène; Taib, Najwa; Debroas, Didier; Domaizon, Isabelle; Jouan Dufournel, Isabelle; Bronner, Gisèle; Salter, Ian; Agogué, Hélène; Mary, Isabelle; Galand, Pierre E

    2013-04-01

    Marine Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, but details regarding their community structure and long-term seasonal activity and dynamics remain largely unexplored. In this study, we monitored the interannual archaeal community composition of abundant and rare biospheres in northwestern Mediterranean Sea surface waters by pyrosequencing 16S rDNA and rRNA. A detailed analysis of the rare biosphere structure showed that the rare archaeal community was composed of three distinct fractions. One contained the rare Archaea that became abundant at different times within the same ecosystem; these cells were typically not dormant, and we hypothesize that they represent a local seed bank that is specific and essential for ecosystem functioning through cycling seasonal environmental conditions. The second fraction contained cells that were uncommon in public databases and not active, consisting of aliens to the studied ecosystem and representing a nonlocal seed bank of potential colonizers. The third fraction contained Archaea that were always rare but actively growing; their affiliation and seasonal dynamics were similar to the abundant microbes and could not be considered a seed bank. We also showed that the major archaeal groups, Thaumarchaeota marine group I and Euryarchaeota group II.B in winter and Euryarchaeota group II.A in summer, contained different ecotypes with varying activities. Our findings suggest that archaeal diversity could be associated with distinct metabolisms or life strategies, and that the rare archaeal biosphere is composed of a complex assortment of organisms with distinct histories that affect their potential for growth.

  8. Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.

    OpenAIRE

    Wettach, J; Gohl, H P; Tschochner, H; Thomm, M

    1995-01-01

    TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show...

  9. A System for Predicting Subcellular Localization of Yeast Genome Using Neural Network

    CERN Document Server

    Thampi, Sabu M

    2007-01-01

    The subcellular location of a protein can provide valuable information about its function. With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important. Many efforts have been made to predict protein subcellular localization. This paper aims to merge the artificial neural networks and bioinformatics to predict the location of protein in yeast genome. We introduce a new subcellular prediction method based on a backpropagation neural network. The results show that the prediction within an error limit of 5 to 10 percentage can be achieved with the system.

  10. Cholinesterase of skeletal muscle and its subcellular components.

    Directory of Open Access Journals (Sweden)

    Fujii,Masafumi

    1982-06-01

    Full Text Available The cholinesterase activity of skeletal muscle and its subcellular components, including motor endplates, was compared chemically in human, mouse and rat. The total cholinesterase activity of muscle per unit protein was in the descending order of human, mouse and rat. Cholinesterase was present in all subcellular components fractionated by differential centrifugation, and was greatest in the microsome fraction followed, in descending order, by the mitochondria, myofibril, and supernatant fractions. Each of these fractions had greater cholinesterase activity in human muscle than in mouse muscle, and in mouse muscle than in rat muscle. The ratio of the activity of the microsome fraction to the activity of muscle homogenate was 11.1 in human, 4.6 in mouse and 3.4 in rat. Because of its relatively greater proportion, the myofibril fraction seems to contribute most to the total cholinesterase activity of muscle. Muscle membrane contained high cholinesterase activity of motor endplates, and the activity was greater than the activity of the microsome fraction in rat. Cholinesterase activity per motor endplate was in the descending order of rat, human and mouse, and the variation was less than the variation in the total muscle cholinesterase activity among these species.

  11. Subcellular Distribution of Glutathione Precursors in Arabidopsis thafiana

    Institute of Scientific and Technical Information of China (English)

    Barbara Eva Koffier; Romana Maier; Bernd Zechmann

    2011-01-01

    Glutathione is an important antioxidant and has many important functions in plant development,growth and defense.Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors,cysteine,glutamate,glycine and y-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis.The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense.The aim of this study was to investigate the compartmentspecific importance of glutathione precursors in Arabidopsis thaliana.The subcellular distribution was compared between wild type plants (Col-0),plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant,wild type plants treated with buthionine sulfoximine),and one complemented line (OE3) with restored glutathione synthesis.Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine,glutamate and glycine in most cell compartments including plastids and the cytosol.A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments.These experiments demonstrated that the inhibition of y-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.

  12. The Vertical Distribution of Sediment Archaeal Community in the “Black Bloom” Disturbing Zhushan Bay of Lake Taihu

    Science.gov (United States)

    Fan, Xianfang; Xing, Peng

    2016-01-01

    Using the Illumina sequencing technology, we investigated the vertical distribution of archaeal community in the sediment of Zhushan Bay of Lake Taihu, where the black bloom frequently occurred in summer. Overall, the Miscellaneous Crenarchaeotal Group (MCG), Deep Sea Hydrothermal Vent Group 6 (DHVEG-6), and Methanobacterium dominated the archaeal community. However, we observed significant difference in composition of archaeal community among different depths of the sediment. DHVEG-6 dominated in the surface layer (0–3 cm) sediment. Methanobacterium was the dominating archaeal taxa in the L2 (3–6 cm) and L3 (6–10) sediment. MCG was most abundant in the L4 (10–15 cm) and L5 (15–20 cm) sediment. Besides, DHVEG-6 was significantly affected by the concentration of total phosphorus (TP). And loss on ignition (LOI) was an important environmental factor for Methanobacterium. As the typical archaeal taxa in the surface layer sediment, DHVEG-6 and Methanobacterium might be more adapted to abundant substrate supply from cyanobacterial blooms and take active part in the biomass transformation. We propose that DHVEG-6 and Methanobacterium could be the key archaeal taxa correlated with the “black bloom” formation in Zhushan Bay. PMID:26884723

  13. Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics.

    Science.gov (United States)

    Evans, Paul N; Parks, Donovan H; Chadwick, Grayson L; Robbins, Steven J; Orphan, Victoria J; Golding, Suzanne D; Tyson, Gene W

    2015-10-23

    Methanogenic and methanotrophic archaea play important roles in the global flux of methane. Culture-independent approaches are providing deeper insight into the diversity and evolution of methane-metabolizing microorganisms, but, until now, no compelling evidence has existed for methane metabolism in archaea outside the phylum Euryarchaeota. We performed metagenomic sequencing of a deep aquifer, recovering two near-complete genomes belonging to the archaeal phylum Bathyarchaeota (formerly known as the Miscellaneous Crenarchaeotal Group). These genomes contain divergent homologs of the genes necessary for methane metabolism, including those that encode the methyl-coenzyme M reductase (MCR) complex. Additional non-euryarchaeotal MCR-encoding genes identified in a range of environments suggest that unrecognized archaeal lineages may also contribute to global methane cycling. These findings indicate that methane metabolism arose before the last common ancestor of the Euryarchaeota and Bathyarchaeota. PMID:26494757

  14. Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics.

    Science.gov (United States)

    Evans, Paul N; Parks, Donovan H; Chadwick, Grayson L; Robbins, Steven J; Orphan, Victoria J; Golding, Suzanne D; Tyson, Gene W

    2015-10-23

    Methanogenic and methanotrophic archaea play important roles in the global flux of methane. Culture-independent approaches are providing deeper insight into the diversity and evolution of methane-metabolizing microorganisms, but, until now, no compelling evidence has existed for methane metabolism in archaea outside the phylum Euryarchaeota. We performed metagenomic sequencing of a deep aquifer, recovering two near-complete genomes belonging to the archaeal phylum Bathyarchaeota (formerly known as the Miscellaneous Crenarchaeotal Group). These genomes contain divergent homologs of the genes necessary for methane metabolism, including those that encode the methyl-coenzyme M reductase (MCR) complex. Additional non-euryarchaeotal MCR-encoding genes identified in a range of environments suggest that unrecognized archaeal lineages may also contribute to global methane cycling. These findings indicate that methane metabolism arose before the last common ancestor of the Euryarchaeota and Bathyarchaeota.

  15. A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

    Science.gov (United States)

    Min, Wonki; Angileri, Francesca; Luo, Haibin; Lauria, Antonino; Shanmugasundaram, Maruda; Almerico, Anna Maria; Cappello, Francesco; de Macario, Everly Conway; Lednev, Igor K; Macario, Alberto J L; Robb, Frank T

    2014-10-27

    Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

  16. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D;

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...... the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that......, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA...

  17. Methanopyrus kandleri: an archaeal methanogen unrelated to all other known methanogens

    Science.gov (United States)

    Burggraf, S.; Stetter, K. O.; Rouviere, P.; Woese, C. R.

    1991-01-01

    Analysis of its 16S rRNA sequence shows that the newly discovered hyperthermophilic methanogen, Methanopryus kandleri, is phylogenetically unrelated to any other known methanogen. The organism represents a separate lineage originating near the root of the archaeal tree. Although the 16S rRNA sequence of Mp. kandleri resembles euryarchaeal 16S rRNAs more than it does crenarchaeal, it shows more crenarchaeal signature features than any known euryarchaeal rRNA. Attempts to place it in relation to the root of the archaeal tree show that the Mp. kandleri lineage likely arises from the euryarchaeal branch of the tree. While the existence of so deeply branching a methanogenic lineage brings into question the thesis that methanogenesis evolved from an earlier metabolism similar to that seen in Thermococcus, it at the same time reinforces the notion that the aboriginal [correction of aborginal] archaeon was a thermophile.

  18. Expression, purification and crystallization of an archaeal-type phosphoenolpyruvate carboxylase

    International Nuclear Information System (INIS)

    The expression, purification, crystallization and preliminary diffraction analysis of an archaeal-type phosphoenolpyruvate carboxylase are described. Complete highly redundant X-ray data have been measured from a crystal diffracting to 3.13 Å resolution. An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in Escherichia coli in a soluble form with an amino-terminal His tag. The recombinant protein is enzymatically active and two crystal forms have been obtained. Complete diffraction data extending to 3.13 Å resolution have been measured from a crystal soaked in KAu(CN)2, using radiation at a wavelength just above the Au LIII edge. The asymmetric unit contains two tetramers of PepcA

  19. Fossilization and degradation of archaeal intact polar tetraether lipids in deeply buried marine sediments (Peru Margin)

    OpenAIRE

    Lengger, S. K.; Hopmans, E.C.; Sinninghe Damsté, J.S.; Schouten, S.

    2014-01-01

    Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX86. GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL-GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death, but it has been suggested that some of these IPL-GDGTs can, just ...

  20. Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

    OpenAIRE

    Gietl, Andreas; Holzmeister, Phil; Blombach, Fabian; Schulz, Sarah; von Voithenberg, Lena Voith; Lamb, Don C; Werner, Finn; Tinnefeld, Philip; Grohmann, Dina

    2014-01-01

    During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP–DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly differen...

  1. Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions

    OpenAIRE

    Hausner, Winfried; Thomm, Michael

    2001-01-01

    Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex s...

  2. Activation of archaeal transcription mediated by recruitment of transcription factor B.

    Science.gov (United States)

    Ochs, Simon M; Thumann, Sybille; Richau, Renate; Weirauch, Matt T; Lowe, Todd M; Thomm, Michael; Hausner, Winfried

    2012-05-25

    Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested. PMID:22496454

  3. Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus▿

    OpenAIRE

    Micorescu, Michael; Grünberg, Sebastian; Franke, Andreas; Cramer, Patrick; Thomm, Michael; Bartlett, Michael

    2007-01-01

    The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription com...

  4. The σ enigma: Bacterial σ factors, archaeal TFB and eukaryotic TFIIB are homologs

    OpenAIRE

    Burton, Samuel P; Burton, Zachary F.

    2014-01-01

    Structural comparisons of initiating RNA polymerase complexes and structure-based amino acid sequence alignments of general transcription initiation factors (eukaryotic TFIIB, archaeal TFB and bacterial σ factors) show that these proteins are homologs. TFIIB and TFB each have two-five-helix cyclin-like repeats (CLRs) that include a C-terminal helix-turn-helix (HTH) motif (CLR/HTH domains). Four homologous HTH motifs are present in bacterial σ factors that are relics of CLR/HTH domains. Sequen...

  5. Factors Controlling the Distribution of Archaeal Tetraethers in Terrestrial Hot Springs▿

    OpenAIRE

    Pearson, Ann; Pi, Yundan; Zhao, Weidong; Li, Wenjun; Li, Yiliang; Inskeep, William; Perevalova, Anna; Romanek, Christopher; Li, Shuguang; Zhang, Chuanlun L.

    2008-01-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, i...

  6. Stratified active archaeal communities in the sediments of Jiulong River Estuary, China

    Directory of Open Access Journals (Sweden)

    Qianqian eLi

    2012-08-01

    Full Text Available Here the composition of total and active archaeal communities in a sediment core of Jiulong River estuary at Fujian Province, Southern China was reported. Profiles of CH4 and SO42- concentrations from the sediment core indicated the existence of a sulfate-methane transition zone (SMTZ in which sulfate reduction-coupled anaerobic oxidation of methane occurs. Accordingly, three sediment layers (16-18.5 cm, 71-73.5 cm, 161-163.5 cm from the 1.2 m sediment core were sectioned and named top, middle and bottom, respectively. Total DNA and RNA of each layer were extracted and used for clone libraries and sequence analysis of 16S rRNA genes, the reverse transcription (RT-PCR products of 16S rRNA and methyl CoM reductase alpha subunit (mcrA genes. Phylogenetic analysis indicated that archaeal communities of the three layers were dominated by the Miscellaneous Crenarchaeotal Group (MCG whose ecological functions were still unknown. The MCG could be further divided into seven subgroups, named MCG-A, B, C, D, E, F and G. MCG-A and MCG-G were the most active groups in the estuarine sediments. Known anaerobic methanotrophic archaea (ANMEs were only found as minor components in these estuarine archaeal communities. This study, together with the studies of deep subsurface sediments, would be a very good start point to target and compare the specific active archaeal groups and their roles in the dark, deep subsurface sediment environments.

  7. Seasonality and resource availability control bacterial and archaeal communities in soils of a temperate beech forest

    OpenAIRE

    Rasche, Frank; Knapp, Daniela; Kaiser, Christina; Koranda, Marianne; Kitzler, Barbara; Zechmeister-Boltenstern, Sophie; Richter, Andreas; Sessitsch, Angela

    2010-01-01

    It was hypothesized that seasonality and resource availability altered through tree girdling were major determinants of the phylogenetic composition of the archaeal and bacterial community in a temperate beech forest soil. During a 2-year field experiment, involving girdling of beech trees to intercept the transfer of easily available carbon (C) from the canopy to roots, members of the dominant phylogenetic microbial phyla residing in top soils under girdled versus untreated control trees wer...

  8. Nitrification of archaeal ammonia oxidizers in a high- temperature hot spring

    Science.gov (United States)

    Chen, Shun; Peng, Xiaotong; Xu, Hengchao; Ta, Kaiwen

    2016-04-01

    The oxidation of ammonia by microbes has been shown to occur in diverse natural environments. However, the link of in situ nitrification activity to taxonomic identities of ammonia oxidizers in high-temperature environments remains poorly understood. Here, we studied in situ ammonia oxidation rates and the diversity of ammonia-oxidizing Archaea (AOA) in surface and bottom sediments at 77 °C in the Gongxiaoshe hot spring, Tengchong, Yunnan, China. The in situ ammonia oxidation rates measured by the 15N-NO3- pool dilution technique in the surface and bottom sediments were 4.80 and 5.30 nmol N g-1 h-1, respectively. Real-time quantitative polymerase chain reaction (qPCR) indicated that the archaeal 16S rRNA genes and amoA genes were present in the range of 0.128 to 1.96 × 108 and 2.75 to 9.80 × 105 gene copies g-1 sediment, respectively, while bacterial amoA was not detected. Phylogenetic analysis of 16S rRNA genes showed high sequence similarity to thermophilic Candidatus Nitrosocaldus yellowstonii, which represented the most abundant operational taxonomic units (OTU) in both surface and bottom sediments. The archaeal predominance was further supported by fluorescence in situ hybridization (FISH) visualization. The cell-specific rate of ammonia oxidation was estimated to range from 0.410 to 0.790 fmol N archaeal cell-1 h-1, higher than those in the two US Great Basin hot springs. These results suggest the importance of archaeal rather than bacterial ammonia oxidation in driving the nitrogen cycle in terrestrial geothermal environments.

  9. Bacterial and archaeal phylogenetic diversity associated with swine sludge from an anaerobic treatment lagoon.

    Science.gov (United States)

    Cardinali-Rezende, Juliana; Pereira, Zelina L; Sanz, José L; Chartone-Souza, Edmar; Nascimento, Andréa M A

    2012-11-01

    Over the last decades, the demand for pork products has increased significantly, along with concern about suitable waste management. Anaerobic-lagoon fermentation for swine-sludge stabilization is a good strategy, although little is known about the microbial communities in the lagoons. Here, we employed a cloning- and sequencing-based analysis of the 16S rRNA gene to characterize and quantify the prokaryotic community composition in a swine-waste-sludge anaerobic lagoon (SAL). DNA sequence analysis revealed that the SAL library harbored 15 bacterial phyla: Bacteroidetes, Cloroflexi, Proteobacteria, Firmicutes, Deinococcus-Thermus, Synergystetes, Gemmatimonadetes, Chlorobi, Fibrobacteres, Verrucomicrobia and candidates division OP5, OP8, WWE1, KSB1, WS6. The SAL library was generally dominated by carbohydrate-oxidizing bacteria. The archaeal sequences were related to the Crenarchaeota and Euryarchaeota phyla. Crenarchaeota predominated in the library, demonstrating that it is not restricted to high-temperature environments, being also responsible for ammonium oxidation in the anaerobic lagoon. Euryarchaeota sequences were associated with the hydrogenotrophic methanogens (Methanomicrobiales and Methanobacteriales). Quantitative PCR analysis revealed that the number of bacterial cells was at least three orders of magnitude higher than the number of archaeal cells in the SAL. The identified prokaryotic diversity was ecologically significant, particularly the archaeal community of hydrogenotrophic methanogens, which was responsible for methane production in the anaerobic lagoon. This study provided insight into the archaeal involvement in the overall oxidation of organic matter and the production of methane. Therefore, the treatment of swine waste in the sludge anaerobic lagoon could represent a potential inoculum for the start-up of municipal solid-waste digesters. PMID:22828793

  10. Significance of archaeal nitrification in hypoxic waters of the Baltic Sea

    OpenAIRE

    Berg, Carlo; Vandieken, Verona; Thamdrup, Bo; Jürgens, Klaus

    2014-01-01

    Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread, and their abundance in many terrestrial and aquatic ecosystems suggests a prominent role in nitrification. AOA also occur in high numbers in oxygen-deficient marine environments, such as the pelagic redox gradients of the central Baltic Sea; however, data on archaeal nitrification rates are scarce and little is known about the factors, for example sulfide, that regulate nitrification in this system. In the present wo...

  11. Temporal changes in soil bacterial and archaeal communities with different fertilizers in tea orchards* #

    OpenAIRE

    Wang, Hua; Yang, Shao-hui; Yang, Jing-ping; Lv, Ya-min; Zhao, Xing; Pang, Ji-liang

    2014-01-01

    It is important to understand the effects of temporal changes in microbial communities in the acidic soils of tea orchards with different fertilizers. A field experiment involving organic fertilizer (OF), chemical fertilizer (CF), and unfertilized control (CK) treatments was arranged to analyze the temporal changes in the bacterial and archaeal communities at bimonthly intervals based on the 16S ribosomal RNA (rRNA) gene using terminal restriction fragment length polymorphism (T-RFLP) profili...

  12. Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea

    OpenAIRE

    Hugoni, Mylène; Agogué, Hélène; Taib, Najwa; Domaizon, Isabelle; Moné, Anne; Pierre E Galand; Bronner, Gisèle; Debroas, Didier; Mary, Isabelle

    2015-01-01

    International audience To test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacte...

  13. Abundance and Composition of Epiphytic Bacterial and Archaeal Ammonia Oxidizers of Marine Red and Brown Macroalgae

    OpenAIRE

    Trias, R. (Rosalía); García-Lledó A. (Arantzazu); Sánchez, N.; López-Jurado, J. L.; Hallin, S. (Sara); Bañeras, Ll. (Lluís)

    2012-01-01

    Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are important for nitrogen cycling in marine ecosystems. Little is known about the diversity and abundance of these organisms on the surface of marine macroalgae, despite the algae’s potential importance to create surfaces and local oxygen-rich environments supporting ammonia oxidation at depths with low dissolved oxygen levels. We determined the abundance and composition of the epiphytic bacterial and archaeal ammonia-oxidizing communities o...

  14. Global Occurrence of Archaeal amoA Genes in Terrestrial Hot Springs▿

    OpenAIRE

    Zhang, Chuanlun L.; Ye, Qi; Huang, Zhiyong; Li, Wenjun; Chen, Jinquan; Song, Zhaoqi; Zhao, Weidong; Bagwell, Christopher; Inskeep, William P.; Ross, Christian; Gao, Lei; Wiegel, Juergen; Romanek, Christopher S.; Shock, Everett L.; Hedlund, Brian P.

    2008-01-01

    Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86°C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (amoA) ...

  15. Biogas production and methanogenic archaeal community in mesophilic and thermophilic anaerobic co-digestion processes.

    Science.gov (United States)

    Yu, D; Kurola, J M; Lähde, K; Kymäläinen, M; Sinkkonen, A; Romantschuk, M

    2014-10-01

    Over 258 Mt of solid waste are generated annually in Europe, a large fraction of which is biowaste. Sewage sludge is another major waste fraction. In this study, biowaste and sewage sludge were co-digested in an anaerobic digestion reactor (30% and 70% of total wet weight, respectively). The purpose was to investigate the biogas production and methanogenic archaeal community composition in the anaerobic digestion reactor under meso- (35-37 °C) and thermophilic (55-57 °C) processes and an increasing organic loading rate (OLR, 1-10 kg VS m(-3) d(-1)), and also to find a feasible compromise between waste treatment capacity and biogas production without causing process instability. In summary, more biogas was produced with all OLRs by the thermophilic process. Both processes showed a limited diversity of the methanogenic archaeal community which was dominated by Methanobacteriales and Methanosarcinales (e.g. Methanosarcina) in both meso- and thermophilic processes. Methanothermobacter was detected as an additional dominant genus in the thermophilic process. In addition to operating temperatures, the OLRs, the acetate concentration, and the presence of key substrates like propionate also affected the methanogenic archaeal community composition. A bacterial cell count 6.25 times higher than archaeal cell count was observed throughout the thermophilic process, while the cell count ratio varied between 0.2 and 8.5 in the mesophilic process. This suggests that the thermophilic process is more stable, but also that the relative abundance between bacteria and archaea can vary without seriously affecting biogas production.

  16. Exploring microbial dark matter to resolve the deep archaeal ancestry of eukaryotes.

    Science.gov (United States)

    Saw, Jimmy H; Spang, Anja; Zaremba-Niedzwiedzka, Katarzyna; Juzokaite, Lina; Dodsworth, Jeremy A; Murugapiran, Senthil K; Colman, Dan R; Takacs-Vesbach, Cristina; Hedlund, Brian P; Guy, Lionel; Ettema, Thijs J G

    2015-09-26

    The origin of eukaryotes represents an enigmatic puzzle, which is still lacking a number of essential pieces. Whereas it is currently accepted that the process of eukaryogenesis involved an interplay between a host cell and an alphaproteobacterial endosymbiont, we currently lack detailed information regarding the identity and nature of these players. A number of studies have provided increasing support for the emergence of the eukaryotic host cell from within the archaeal domain of life, displaying a specific affiliation with the archaeal TACK superphylum. Recent studies have shown that genomic exploration of yet-uncultivated archaea, the so-called archaeal 'dark matter', is able to provide unprecedented insights into the process of eukaryogenesis. Here, we provide an overview of state-of-the-art cultivation-independent approaches, and demonstrate how these methods were used to obtain draft genome sequences of several novel members of the TACK superphylum, including Lokiarchaeum, two representatives of the Miscellaneous Crenarchaeotal Group (Bathyarchaeota), and a Korarchaeum-related lineage. The maturation of cultivation-independent genomics approaches, as well as future developments in next-generation sequencing technologies, will revolutionize our current view of microbial evolution and diversity, and provide profound new insights into the early evolution of life, including the enigmatic origin of the eukaryotic cell. PMID:26323759

  17. Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China.

    Science.gov (United States)

    Song, Zhao-Qi; Wang, Feng-Ping; Zhi, Xiao-Yang; Chen, Jin-Quan; Zhou, En-Min; Liang, Feng; Xiao, Xiang; Tang, Shu-Kun; Jiang, Hong-Chen; Zhang, Chuanlun L; Dong, Hailiang; Li, Wen-Jun

    2013-04-01

    Thousands of hot springs are located in the north-eastern part of the Yunnan-Tibet geothermal zone, which is one of the most active geothermal areas in the world. However, a comprehensive and detailed understanding of microbial diversity in these hot springs is still lacking. In this study, bacterial and archaeal diversities were investigated in 16 hot springs (pH 3.2-8.6; temperature 47-96°C) in Yunnan Province and Tibet, China by using a barcoded 16S rRNA gene-pyrosequencing approach. Aquificae, Proteobacteria, Firmicutes, Deinococcus-Thermus and Bacteroidetes comprised the large portion of the bacterial communities in acidic hot springs. Non-acidic hot springs harboured more and variable bacterial phyla than acidic springs. Desulfurococcales and unclassified Crenarchaeota were the dominated groups in archaeal populations from most of the non-acidic hot springs; whereas, the archaeal community structure in acidic hot springs was simpler and characterized by Sulfolobales and Thermoplasmata. The phylogenetic analyses showed that Aquificae and Crenarchaeota were predominant in the investigated springs and possessed many phylogenetic lineages that have never been detected in other hot springs in the world. Thus findings from this study significantly improve our understanding of microbial diversity in terrestrial hot springs.

  18. Archaeal community structures in the solfataric acidic hot springs with different temperatures and elemental compositions.

    Science.gov (United States)

    Satoh, Tomoko; Watanabe, Keiko; Yamamoto, Hideo; Yamamoto, Shuichi; Kurosawa, Norio

    2013-01-01

    Archaeal 16S rRNA gene compositions and environmental factors of four distinct solfataric acidic hot springs in Kirishima, Japan were compared. The four ponds were selected by differences of temperature and total dissolved elemental concentration as follows: (1) Pond-A: 93°C and 1679 mg L(-1), (2) Pond-B: 66°C and 2248 mg L(-1), (3) Pond-C: 88°C and 198 mg L(-1), and (4) Pond-D: 67°C and 340 mg L(-1). In total, 431 clones of 16S rRNA gene were classified into 26 phylotypes. In Pond-B, the archaeal diversity was the highest among the four, and the members of the order Sulfolobales were dominant. The Pond-D also showed relatively high diversity, and the most frequent group was uncultured thermoacidic spring clone group. In contrast to Pond-B and Pond-D, much less diverse archaeal clones were detected in Pond-A and Pond-C showing higher temperatures. However, dominant groups in these ponds were also different from each other. The members of the order Sulfolobales shared 89% of total clones in Pond-A, and the uncultured crenarchaeal groups shared 99% of total Pond-C clones. Therefore, species compositions and biodiversity were clearly different among the ponds showing different temperatures and dissolved elemental concentrations.

  19. Archaeal Community Changes Associated with Cultivation of Amazon Forest Soil with Oil Palm.

    Science.gov (United States)

    Tupinambá, Daiva Domenech; Cantão, Maurício Egídio; Costa, Ohana Yonara Assis; Bergmann, Jessica Carvalho; Kruger, Ricardo Henrique; Kyaw, Cynthia Maria; Barreto, Cristine Chaves; Quirino, Betania Ferraz

    2016-01-01

    This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied.

  20. Assessment of bacterial and archaeal community structure in Swine wastewater treatment processes.

    Science.gov (United States)

    Da Silva, Marcio Luis Busi; Cantão, Mauricio Egídio; Mezzari, Melissa Paola; Ma, Jie; Nossa, Carlos Wolfgang

    2015-07-01

    Microbial communities from two field-scale swine wastewater treatment plants (WWTPs) were assessed by pyrosequencing analyses of bacterial and archaeal 16S ribosomal DNA (rDNA) fragments. Effluent samples from secondary (anaerobic covered lagoons and upflow anaerobic sludge blanket [UASB]) and tertiary treatment systems (open-pond natural attenuation lagoon and air-sparged nitrification-denitrification tank followed by alkaline phosphorus precipitation process) were analyzed. A total of 56,807 and 48,859 high-quality reads were obtained from bacterial and archaeal libraries, respectively. Dominant bacterial communities were associated with the phylum Firmicutes, Bacteroidetes, Proteobacteria, or Actinobacteria. Bacteria and archaea diversity were highest in UASB effluent sample. Escherichia, Lactobacillus, Bacteroides, and/or Prevotella were used as indicators of putative pathogen reduction throughout the WWTPs. Satisfactory pathogen reduction was observed after the open-pond natural attenuation lagoon but not after the air-sparged nitrification/denitrification followed by alkaline phosphorus precipitation treatment processes. Among the archaeal communities, 80% of the reads was related to hydrogeno-trophic methanogens Methanospirillum. Enrichment of hydrogenotrophic methanogens detected in effluent samples from the anaerobic covered lagoons and UASB suggested that CO2 reduction with H2 was the dominant methanogenic pathway in these systems. Overall, the results served to improve our current understanding of major microbial communities' changes downgradient from the pen and throughout swine WWTP as a result of different treatment processes. PMID:25432577

  1. Archaeal Community Changes Associated with Cultivation of Amazon Forest Soil with Oil Palm

    Science.gov (United States)

    Tupinambá, Daiva Domenech; Cantão, Maurício Egídio; Costa, Ohana Yonara Assis; Bergmann, Jessica Carvalho; Kruger, Ricardo Henrique; Kyaw, Cynthia Maria; Barreto, Cristine Chaves; Quirino, Betania Ferraz

    2016-01-01

    This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied. PMID:27006640

  2. Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea.

    Science.gov (United States)

    Hugoni, Mylène; Agogué, Hélène; Taib, Najwa; Domaizon, Isabelle; Moné, Anne; Galand, Pierre E; Bronner, Gisèle; Debroas, Didier; Mary, Isabelle

    2015-08-01

    To test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacterial amoA transcripts. Our results showed a succession of different nitrifiers from river to sea with bacterial amoA transcripts dominating in the freshwater station while archaeal transcripts were predominant in the marine station. The 16S rRNA sequence analysis revealed that Thaumarchaeota marine group I (MGI) were the most abundant overall but other archaeal groups like Methanosaeta were also potentially active in winter (December-March) and Euryarchaeota marine group II (MGII) were dominant in seawater in summer (April-August). Each station also contained different Thaumarchaeota MGI phylogenetic clusters, and the clusters' microdiversity was associated to specific environmental conditions suggesting the presence of ecotypes adapted to distinct ecological niches. The amoA and ureC transcript dynamics further indicated that some of the Thaumarchaeota MGI subclusters were involved in ammonia oxidation through the hydrolysis of urea. Our findings show that ammonia-oxidizing Archaea and Bacteria were adapted to contrasted conditions and that the Thaumarchaeota MGI diversity probably corresponds to distinct metabolisms or life strategies. PMID:25851445

  3. Archaeal Community Changes Associated with Cultivation of Amazon Forest Soil with Oil Palm

    Directory of Open Access Journals (Sweden)

    Daiva Domenech Tupinambá

    2016-01-01

    Full Text Available This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7% were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area. More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied.

  4. An image analysis method to quantify CFTR subcellular localization.

    Science.gov (United States)

    Pizzo, Lucilla; Fariello, María Inés; Lepanto, Paola; Aguilar, Pablo S; Kierbel, Arlinet

    2014-08-01

    Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify

  5. Microscopy with spatial filtering for monitoring subcellular morphology

    Science.gov (United States)

    Zheng, Jing-Yi

    Dynamic alteration in organelle morphology is an important indicator of cellular function and many efforts have been made to monitor the subcellular morphology. Optical scatter imaging (OSI), which combines light scattering spectroscopy with microscopic imaging, was developed to non-invasively track real-time changes in particle morphology in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter (OSIR, optical scatter imaging ratio) at each pixel in the full field of view. For spherical particles, the OSIR was shown to decrease monotonically with diameter. In living cells, we reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl- xL transmembrane domain, but could not be observed by fluorescence or DIC images1. However, the initial design was based on Mie theory of scattering by spheres, and hence only adequate for measuring spherical particles. This limits the applicability of OSI to cellular functional studies involving organelles, which are naturally non-spherical. In this project, we aim to enhance the current capability of the existing optical scatter microscope to assess size and shape information for both spherical and non-spherical particles, and eventually apply this technique for monitoring and quantifying subcellular morphology within living cells. To reach this goal, we developed an improved system, in which the variable diameter iris is replaced with a digital micromirror device and adopted the concept of Gabor filtering to extend our assessment of morphology to the characterization of particle shape and orientation. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This

  6. Characterization of an archaeal two-component system that regulates methanogenesis in Methanosaeta harundinacea.

    Directory of Open Access Journals (Sweden)

    Jie Li

    Full Text Available Two-component signal transduction systems (TCSs are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase and its cognate response regulator. FilR1 carries a receiver (REC domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.

  7. Functional analysis of archaeal MBF1 by complementation studies in yeast

    Directory of Open Access Journals (Sweden)

    Siebers Bettina

    2011-03-01

    Full Text Available Abstract Background Multiprotein-bridging factor 1 (MBF1 is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP like proteins (e.g. Gcn4 in yeast or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect and the TATA-box binding protein (TBP in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1 and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ as well as domain swap experiments between aMBF1 and yMbf1 were performed. Results In contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ. Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1. However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus

  8. Imaging protein interactions in vivo with sub-cellular resolution

    CERN Document Server

    Raicu, Valerica; Fung, Russell; Melnichuk, Mike; Jansma, David B; Pisterzi, Luca; Fox, Michael; Wells, James W; Saldin, Dilano K

    2008-01-01

    Resonant Energy Transfer (RET) from an optically excited donor molecule (D) to a non-excited acceptor molecule (A) residing nearby is widely used to detect molecular interactions in living cells. Stoichiometric information, such as the number of proteins forming a complex, has been obtained so far for a handful of proteins, but only after exposing the sample sequentially to at least two different excitation wavelengths. During this lengthy process of measurement, the molecular makeup of a cellular region may change, and this has so far limited the applicability of RET to determination of cellular averages. Here we demonstrate a method for imaging protein complex distribution in living cells with sub-cellular spatial resolution, which relies on a spectrally-resolved two-photon microscope, a simple but competent theory, and a keen selection of fluorescent tags. This technology may eventually lead to tracking dynamics of macromolecular complex formation and dissociation with spatial resolution inside living cell...

  9. Torso RTK controls Capicua degradation by changing its subcellular localization.

    Science.gov (United States)

    Grimm, Oliver; Sanchez Zini, Victoria; Kim, Yoosik; Casanova, Jordi; Shvartsman, Stanislav Y; Wieschaus, Eric

    2012-11-01

    The transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts. PMID:23048183

  10. Quantitative Dose-Response Curves from Subcellular Lipid Multilayer Microarrays

    Science.gov (United States)

    Kusi-Appiah, A. E.; Lowry, T. W.; Darrow, E. M.; Wilson, K.; Chadwick, B. P.; Davidson, M. W.; Lenhert, S.

    2015-01-01

    The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate. PMID:26167949

  11. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Directory of Open Access Journals (Sweden)

    Qian-Quan Sun

    Full Text Available To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2 is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites. We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  12. Spatiotemporal dynamics of bacterial and archaeal communities in household biogas digesters from tropical and subtropical regions of Yunnan Province, China.

    Science.gov (United States)

    Tian, Guangliang; Li, Qiumin; Dong, Minghua; Wu, Yan; Yang, Bin; Zhang, Lijuan; Li, Yingjuan; Yin, Fang; Zhao, Xingling; Wang, Yongxia; Xiao, Wei; Cui, Xiaolong; Zhang, Wudi

    2016-06-01

    A combination of 16S rRNA gene PCR-based techniques and the determination of abiotic factors were used to study community composition, richness, and evenness and the correlation between biotic and abiotic factors in 19 household biogas digesters in tropical and subtropical regions of Yunnan Province, China. The results revealed that both bacterial and archaeal community composition differed between regions and archaeal community composition was more affected by season than bacterial; regardless of sampling location, the dominant bacterial phyla included Chloroflexi, Bacteroidetes, Firmicutes, and Proteobacteria, and the most dominant archaeal phylum was Euryarchaeota; in digesters from both regions, Chloroflexi as the first or second most dominant bacteria accounted for 21.50-26.10 % of bacterial library sequences, and the phylum Crenarchaeota as the second most dominant archaea accounted for 17.65-19.77 % of archaeal library sequences; the species Methanosaeta concilii as the most dominant archaeal species accounted for 67.80-72.80 % of the sequences. This study found that most of the abundant microbial communities in 19 biogas digesters are similar, and this result will provide enlightenment for finding the universal nature in rural biogas digesters at tropical and subtropical regions in China. PMID:26916266

  13. Bacterial and archaeal communities in the deep-sea sediments of inactive hydrothermal vents in the Southwest India Ridge.

    Science.gov (United States)

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Xu, Jian; Shuai, Yinjie; Zhou, Xiaojian; Yang, Zhihui; Ma, Kesen

    2016-01-01

    Active deep-sea hydrothermal vents harbor abundant thermophilic and hyperthermophilic microorganisms. However, microbial communities in inactive hydrothermal vents have not been well documented. Here, we investigated bacterial and archaeal communities in the two deep-sea sediments (named as TVG4 and TVG11) collected from inactive hydrothermal vents in the Southwest India Ridge using the high-throughput sequencing technology of Illumina MiSeq2500 platform. Based on the V4 region of 16S rRNA gene, sequence analysis showed that bacterial communities in the two samples were dominated by Proteobacteria, followed by Bacteroidetes, Actinobacteria and Firmicutes. Furthermore, archaeal communities in the two samples were dominated by Thaumarchaeota and Euryarchaeota. Comparative analysis showed that (i) TVG4 displayed the higher bacterial richness and lower archaeal richness than TVG11; (ii) the two samples had more divergence in archaeal communities than bacterial communities. Bacteria and archaea that are potentially associated with nitrogen, sulfur metal and methane cycling were detected in the two samples. Overall, we first provided a comparative picture of bacterial and archaeal communities and revealed their potentially ecological roles in the deep-sea environments of inactive hydrothermal vents in the Southwest Indian Ridge, augmenting microbial communities in inactive hydrothermal vents.

  14. Bacterial and archaeal communities in the deep-sea sediments of inactive hydrothermal vents in the Southwest India Ridge

    Science.gov (United States)

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Xu, Jian; Shuai, Yinjie; Zhou, Xiaojian; Yang, Zhihui; Ma, Kesen

    2016-05-01

    Active deep-sea hydrothermal vents harbor abundant thermophilic and hyperthermophilic microorganisms. However, microbial communities in inactive hydrothermal vents have not been well documented. Here, we investigated bacterial and archaeal communities in the two deep-sea sediments (named as TVG4 and TVG11) collected from inactive hydrothermal vents in the Southwest India Ridge using the high-throughput sequencing technology of Illumina MiSeq2500 platform. Based on the V4 region of 16S rRNA gene, sequence analysis showed that bacterial communities in the two samples were dominated by Proteobacteria, followed by Bacteroidetes, Actinobacteria and Firmicutes. Furthermore, archaeal communities in the two samples were dominated by Thaumarchaeota and Euryarchaeota. Comparative analysis showed that (i) TVG4 displayed the higher bacterial richness and lower archaeal richness than TVG11; (ii) the two samples had more divergence in archaeal communities than bacterial communities. Bacteria and archaea that are potentially associated with nitrogen, sulfur metal and methane cycling were detected in the two samples. Overall, we first provided a comparative picture of bacterial and archaeal communities and revealed their potentially ecological roles in the deep-sea environments of inactive hydrothermal vents in the Southwest Indian Ridge, augmenting microbial communities in inactive hydrothermal vents.

  15. Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

    Science.gov (United States)

    Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

    2012-08-01

    Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

  16. Factors Controlling the Distribution of Archaeal Tetraethers in Terrestrial Hot Springs▿

    Science.gov (United States)

    Pearson, Ann; Pi, Yundan; Zhao, Weidong; Li, WenJun; Li, Yiliang; Inskeep, William; Perevalova, Anna; Romanek, Christopher; Li, Shuguang; Zhang, Chuanlun L.

    2008-01-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX86 paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies. PMID:18390673

  17. Factors controlling the distribution of archaeal tetraethers in terrestrial hot springs.

    Science.gov (United States)

    Pearson, Ann; Pi, Yundan; Zhao, Weidong; Li, WenJun; Li, Yiliang; Inskeep, William; Perevalova, Anna; Romanek, Christopher; Li, Shuguang; Zhang, Chuanlun L

    2008-06-01

    Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87 degrees C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX(86) paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies. PMID:18390673

  18. Bacterial and Archaeal Diversity in the Gastrointestinal Tract of the North American Beaver (Castor canadensis)

    Science.gov (United States)

    Gruninger, Robert J.; McAllister, Tim A.; Forster, Robert J.

    2016-01-01

    The North American Beaver (Castor canadensis) is the second largest living rodent and an iconic symbol of Canada. The beaver is a semi-aquatic browser whose diet consists of lignocellulose from a variety of plants. The beaver is a hindgut fermenter and has an enlarged ceacum that houses a complex microbiome. There have been few studies examining the microbial diversity in gastrointestinal tract of hindgut fermenting herbivores. To examine the bacterial and archaeal communities inhabiting the gastrointestinal tract of the beaver, the microbiome of the ceacum and feaces was examined using culture-independent methods. DNA from the microbial community of the ceacum and feaces of 4 adult beavers was extracted, and the16S rRNA gene was sequenced using either bacterial or archaeal specific primers. A total of 1447 and 1435 unique bacterial OTUs were sequenced from the ceacum and feaces, respectively. On average, the majority of OTUs within the ceacum were classified as Bacteroidetes (49.2%) and Firmicutes (47.6%). The feaces was also dominated by OTUs from Bacteroidetes (36.8%) and Firmicutes (58.9%). The composition of bacterial community was not significantly different among animals. The composition of the ceacal and feacal microbiome differed, but this difference is due to changes in the abundance of closely related OTUs, not because of major differences in the taxonomic composition of the communities. Within these communities, known degraders of lignocellulose were identified. In contrast, to the bacterial microbiome, the archaeal community was dominated by a single species of methanogen, Methanosphaera stadtmanae. The data presented here provide the first insight into the microbial community within the hindgut of the beaver. PMID:27227334

  19. Plant genotype-specific archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive trees

    Directory of Open Access Journals (Sweden)

    Henry eMueller

    2015-03-01

    Full Text Available Endophytes have an intimate and often symbiotic interaction with their hosts. Less is known about the composition and function of endophytes in trees. In order to evaluate our hypothesis that plant genotype and origin have a strong impact on both, endophytes of leaves from 10 Olea europaea L. cultivars from the Mediterranean basin growing at a single agricultural site in Spain and from nine wild olive trees located in natural habitats in Greece, Cyprus and on Madeira Island were studied. The composition of the bacterial endophytic communities as revealed by 16S rRNA gene amplicon sequencing and the subsequent PCoA analysis showed a strong correlation to the plant genotypes. The bacterial distribution patterns were congruent with the plant origins in Eastern and Western areas of the Mediterranean basin. Subsequently, the endophytic microbiome of wild olives was shown to be closely related to those of cultivated olives of the corresponding geographic origins. The olive leaf endosphere harbored mostly Proteobacteria, followed by Firmicutes, Actinobacteria and Bacteroidetes. The detection of a high portion of archaeal taxa belonging to the phyla Thaumarchaeota, Crenarchaeota and Euryarchaeota in the amplicon libraries was an unexpected discovery, which was confirmed by quantitative real-time PCR revealing an archaeal portion of up to 35.8%. Although the function of these Archaea for their host plant remains speculative, this finding suggests a significant relevance of archaeal endophytes for plant-microbe interactions. In addition, the antagonistic potential of culturable endophytes was determined; all isolates with antagonistic activity against the olive-pathogenic fungus Verticillium dahliae Kleb. belong to Bacillus amyloliquefaciens. In contrast to the specific global structural diversity, BOX-fingerprints of the antagonistic Bacillus isolates were highly similar and independent of the olive genotype from which they were isolated.

  20. Land-use systems affect Archaeal community structure and functional diversity in western Amazon soils

    Directory of Open Access Journals (Sweden)

    Acácio Aparecido Navarrete

    2011-10-01

    Full Text Available The study of the ecology of soil microbial communities at relevant spatial scales is primordial in the wide Amazon region due to the current land use changes. In this study, the diversity of the Archaea domain (community structure and ammonia-oxidizing Archaea (richness and community composition were investigated using molecular biology-based techniques in different land-use systems in western Amazonia, Brazil. Soil samples were collected in two periods with high precipitation (March 2008 and January 2009 from Inceptisols under primary tropical rainforest, secondary forest (5-20 year old, agricultural systems of indigenous people and cattle pasture. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE using the 16S rRNA gene as a biomarker showed that archaeal community structures in crops and pasture soils are different from those in primary forest soil, which is more similar to the community structure in secondary forest soil. Sequence analysis of excised DGGE bands indicated the presence of crenarchaeal and euryarchaeal organisms. Based on clone library analysis of the gene coding the subunit of the enzyme ammonia monooxygenase (amoA of Archaea (306 sequences, the Shannon-Wiener function and Simpson's index showed a greater ammonia-oxidizing archaeal diversity in primary forest soils (H' = 2.1486; D = 0.1366, followed by a lower diversity in soils under pasture (H' = 1.9629; D = 0.1715, crops (H' = 1.4613; D = 0.3309 and secondary forest (H' = 0.8633; D = 0.5405. All cloned inserts were similar to the Crenarchaeota amoA gene clones (identity > 95 % previously found in soils and sediments and distributed primarily in three major phylogenetic clusters. The findings indicate that agricultural systems of indigenous people and cattle pasture affect the archaeal community structure and diversity of ammonia-oxidizing Archaea in western Amazon soils.

  1. Seasonal dynamics of bacterial and archaeal methanogenic communities in flooded rice fields and effect of drainage

    Directory of Open Access Journals (Sweden)

    Björn eBreidenbach

    2015-01-01

    Full Text Available We studied the resident (16S rDNA and the active (16S rRNA members of soil archaeal and bacterial communities during rice plant development by sampling three growth stages (vegetative, reproductive and maturity under field conditions. Additionally, the microbial community was investigated in two non-flooded fields (unplanted, cultivated with upland maize in order to monitor the reaction of the microbial communities to non-flooded, dry conditions. The abundance of Bacteria and Archaea was monitored by quantitative PCR showing an increase in 16S rDNA during reproductive stage and stable 16S rRNA copies throughout the growth season. Community profiling by T-RFLP indicated a relatively stable composition during rice plant growth whereas pyrosequencing revealed minor changes in relative abundance of a few bacterial groups. Comparison of the two non-flooded fields with flooded rice fields showed that the community composition of the Bacteria was slightly different, while that of the Archaea was almost the same. Only the relative abundance of Methanosarcinaceae and Soil Crenarchaeotic Group increased in non-flooded versus flooded soil. The abundance of bacterial and archaeal 16S rDNA copies was highest in flooded rice fields, followed by non-flooded maize and unplanted fields. However, the abundance of ribosomal RNA (active microbes was similar indicating maintenance of a high level of ribosomal RNA under the non-flooded conditions, which were unfavorable for anaerobic bacteria and methanogenic archaea. This maintenance possibly serves as preparedness for activity when conditions improve. In summary, the analyses showed that the bacterial and archaeal communities inhabiting Philippine rice field soil were relatively stable over the season but reacted upon change in field management.

  2. Response of Archaeal and Bacterial Soil Communities to Changes Associated with Outdoor Cattle Overwintering.

    Directory of Open Access Journals (Sweden)

    Alica Chroňáková

    Full Text Available Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of four Czech grassland soils affected by outdoor cattle husbandry. Two show short-term (3 years; STI and long-term impact (17 years; LTI, one is regenerating from cattle impact (REG and a control is unaffected by cattle (CON. Cattle manure (CMN, the source of allochthonous microbes, was collected from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition of archaeal and bacterial communities in each soil type and CMN. Both short- and long- term cattle impact negatively altered archaeal and bacterial diversity, leading to increase of homogenization of microbial communities in overwintering soils over time. Moreover, strong shifts in the prokaryotic communities were observed in response to cattle overwintering, with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g. Thaumarchaeota, Acidobacteria, and α-Proteobacteria dominated in CON and expressed strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria, and Bacteroidetes were common in LTI showing opposite trends. Crenarchaeota were also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes, Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae suggested increased abundance of soil-borne microbes under altered environmental conditions. The observed changes in prokaryotic community composition may have driven corresponding changes in soil functioning.

  3. Diversity of putative archaeal RNA viruses in metagenomic datasets of a yellowstone acidic hot spring.

    OpenAIRE

    Hongming WANG; Yu, Yongxin; Liu, Taigang; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2015-01-01

    Two genomic fragments (5,662 and 1,269 nt in size, GenBank accession no. JQ756122 and JQ756123, respectively) of novel, positive-strand RNA viruses that infect archaea were first discovered in an acidic hot spring in Yellowstone National Park (Bolduc et al., 2012). To investigate the diversity of these newly identified putative archaeal RNA viruses, global metagenomic datasets were searched for sequences that were significantly similar to those of the viruses. A total of 3,757 associated read...

  4. Effect of Tree Species and Mycorrhizal Colonization on the Archaeal Population of Boreal Forest Rhizospheres▿

    OpenAIRE

    Bomberg, Malin; Timonen, Sari

    2008-01-01

    Group 1.1c Crenarchaeota are the predominating archaeal group in acidic boreal forest soils. In this study, we show that the detection frequency of 1.1c crenarchaeotal 16S rRNA genes in the rhizospheres of the boreal forest trees increased following colonization by the ectomycorrhizal fungus Paxillus involutus. This effect was very clear in the fine roots of Pinus sylvestris, Picea abies, and Betula pendula, the most common forest trees in Finland. The nonmycorrhizal fine roots had a clearly ...

  5. Seasonal Effects in a Lake Sediment Archaeal Community of the Brazilian Savanna

    OpenAIRE

    Thiago Rodrigues; Elisa Catão; Mercedes M. C. Bustamante; Quirino, Betania F.; Kruger, Ricardo H; Kyaw, Cynthia M

    2014-01-01

    The Cerrado is a biome that corresponds to 24% of Brazil’s territory. Only recently microbial communities of this biome have been investigated. Here we describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerrado in the dry season and in the transition period between the dry and rainy seasons, when the first rains occur. Gene libraries were constructed, using Archaea-specific primers for the 16S rRNA and amoA genes. Analysis revealed marked ...

  6. Protein-protein interaction as a predictor of subcellular location

    Directory of Open Access Journals (Sweden)

    Davis Melissa J

    2009-02-01

    Full Text Available Abstract Background Many biological processes are mediated by dynamic interactions between and among proteins. In order to interact, two proteins must co-occur spatially and temporally. As protein-protein interactions (PPIs and subcellular location (SCL are discovered via separate empirical approaches, PPI and SCL annotations are independent and might complement each other in helping us to understand the role of individual proteins in cellular networks. We expect reliable PPI annotations to show that proteins interacting in vivo are co-located in the same cellular compartment. Our goal here is to evaluate the potential of using PPI annotation in determining SCL of proteins in human, mouse, fly and yeast, and to identify and quantify the factors that contribute to this complementarity. Results Using publicly available data, we evaluate the hypothesis that interacting proteins must be co-located within the same subcellular compartment. Based on a large, manually curated PPI dataset, we demonstrate that a substantial proportion of interacting proteins are in fact co-located. We develop an approach to predict the SCL of a protein based on the SCL of its interaction partners, given sufficient confidence in the interaction itself. The frequency of false positive PPIs can be reduced by use of six lines of supporting evidence, three based on type of recorded evidence (empirical approach, multiplicity of databases, and multiplicity of literature citations and three based on type of biological evidence (inferred biological process, domain-domain interactions, and orthology relationships, with biological evidence more-effective than recorded evidence. Our approach performs better than four existing prediction methods in identifying the SCL of membrane proteins, and as well as or better for soluble proteins. Conclusion Understanding cellular systems requires knowledge of the SCL of interacting proteins. We show how PPI data can be used more effectively to

  7. Changes in archaeal abundance and community structure along a salinity gradient in the lower Pearl River and its estuary

    Science.gov (United States)

    Zhang, C.; Wang, J.; Xie, W.; Wang, P.; Wei, Y.; Chen, S.; Zhou, X.

    2013-12-01

    Archaea occur in a wide range of habitats and across broad environmental gradients. At the global scale, salinity is known to be a major driving force for archaeal species diversity. The goal of this study was to examine changes in abundance and diversity of archaeal community DNA and membrane lipids in the water column along a salinity gradient in the lower Pearl River and estuary in the context of water/gas chemistry (pH, nitrate/nitrite, ammonia, methane, carbon dioxide). The pH increased and nitrate/nitrite and ammonia decreased from the lower Pearl River to the estuary. Methane and carbon dioxide fluxes were high in the lower Pearl River and decreased sharply in the estuary and toward the open ocean. The archaeal lipid profile exhibited abrupt changes from dominance of GDGT-0 (a glycerol diakly glycerol tetraether with zero cyclopentyl ring, which is commonly present in methanogens) to dominance of crenarchaeol (a specific biomarker for Thaumarchaeota) with increasing salinity from zero in the lower Pearl River to >0.5% in the estuary. Quantification of the 16S rRNA gene abundance using qPCR revealed a switch from bacteria-dominance to archaea-dominance and the ratio of archaeal nirK/bacterial-amoA genes had a peak value in the estuary, suggesting enhanced activity of ammonia oxidation by archaea. Pyrosequencing of archaeal 16S rRNA, amoA and nirK genes exhibited systematic variation defined by habitat types. Our current studies employ rate measurements of carbon fixation, ammonia oxidation, and nitrate reduction using isotope labeling approaches, which will allow us to link changes in archaeal community structure and ecological function.

  8. Subcellular localization of rickettsial invasion protein, InvA.

    Science.gov (United States)

    Gaywee, Jariyanart; Sacci, John B; Radulovic, Suzana; Beier, Magda S; Azad, Abdu F

    2003-01-01

    To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

  9. Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

    Institute of Scientific and Technical Information of China (English)

    Juan TAN; Kai WU; Rui CHANG; Qi-min CHEN; Yun-qi GENG; Wen-tao QIAO

    2008-01-01

    The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.

  10. Global, quantitative and dynamic mapping of protein subcellular localization

    Science.gov (United States)

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  11. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

    KAUST Repository

    Bayoumi, Maged Fouad

    2012-12-12

    To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards. © 2012 American Chemical Society.

  12. Subcellular and in-vivo Nano-Endoscopy

    Science.gov (United States)

    Cheemalapati, Surya Venkatasekhar; Winskas, John; Wang, Hao; Konnaiyan, Karthik; Zhdanov, Arseny; Roth, Alison; Adapa, Swamy Rakesh; Deonarine, Andrew; Noble, Mark; Das, Tuhin; Gatenby, Robert; Westerheide, Sandy D.; Jiang, Rays H. Y.; Pyayt, Anna

    2016-01-01

    Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level. PMID:27694854

  13. Correction: Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    Science.gov (United States)

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  14. Physiological aspects of the subcellular localization of glycogen in skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Ørtenblad, Niels

    2013-01-01

    with metabolic and scaffolding proteins. Although the subcellular localization of glycogen has been recognized for more than 40 years, the physiological role of the distinct localizations has received sparse attention. Recently, however, studies involving stereological, unbiased, quantitative methods have...

  15. Environmental and Genetic Influences of Archaeal Lipid Distribution in Natural and Artificial Marine Environments

    Science.gov (United States)

    Warren, C.; Pagani, M.

    2012-12-01

    TEX86 is a proxy of sea surface temperature based on refractory glycerol dibiphytanyl glycerol tetraethers (GDGT) in the cell membranes of low-temperature dwelling (non-hyperthermophilic) Archaea. The degree to which environmental signals other than temperature influence the distribution of GDGT compounds is poorly understood. Few representatives of the Thaumarchaeota — the clade to which the dominant GDGT production has been attributed — have been described or isolated in pure culture, and the role of genetic lineage in the synthesis and distribution of GDGTs is unknown. For this project we collected water, filter and substrate samples from tank systems in non-profit and commercial aquariums around the United States. This analysis compares GDGT core lipids and intact polar lipid distributions with Archaeal genetic sequence data processed using rRNA and 454 Pyrosequencing. Environmental attributes (such as dissolved oxygen concentration, salinity, organic density, etc.) specific to each tank are also compared to lipid analyses and the presence of specific lineages within select tank systems. Our preliminary results demonstrate that archaeal GDGTs are present and abundant within a range of environmental conditions, including artificial saline and brackish waters derived from municipal sources. Comparisons of existing TEX86 calibration values with known temperatures suggest that residuals vary based on non-temperature parameters. Branched compounds are absent in most aquarium systems, but dominate in systems prepared with municipal water.

  16. Archaeal and bacterial diversity in acidic to circumneutral hot springs in the Philippines.

    Science.gov (United States)

    Huang, Qiuyuan; Jiang, Hongchen; Briggs, Brandon R; Wang, Shang; Hou, Weiguo; Li, Gaoyuan; Wu, Geng; Solis, Ramonito; Arcilla, Carlo A; Abrajano, Teofilo; Dong, Hailiang

    2013-09-01

    The microbial diversity was investigated in sediments of six acidic to circumneutral hot springs (Temperature: 60-92 °C, pH 3.72-6.58) in the Philippines using an integrated approach that included geochemistry and 16S rRNA gene pyrosequencing. Both bacterial and archaeal abundances were lower in high-temperature springs than in moderate-temperature ones. Overall, the archaeal community consisted of sequence reads that exhibited a high similarity (nucleotide identity > 92%) to phyla Crenarchaeota, Euryarchaeota, and unclassified Archaea. The bacterial community was composed of sequence reads moderately related (nucleotide identity > 90%) to 17 phyla, with Aquificae and Firmicutes being dominant. These phylogenetic groups were correlated with environmental conditions such as temperature, dissolved sulfate and calcium concentrations in spring water, and sediment properties including total nitrogen, pyrite, and elemental sulfur. Based on the phylogenetic inference, sulfur metabolisms appear to be key physiological functions in these hot springs. Sulfobacillus (within phylum Firmicutes) along with members within Sulfolobales were abundant in two high-temperature springs (> 76 °C), and they were hypothesized to play an important role in regulating the sulfur cycling under high-temperature conditions. The results of this study improve our understanding of microbial diversity and community composition in acidic to circumneutral terrestrial hot springs and their relationships with geochemical conditions.

  17. Archaeal Populations in Hypersaline Sediments Underlying Orange Microbial Mats in the Napoli Mud Volcano▿†

    Science.gov (United States)

    Lazar, Cassandre Sara; L'Haridon, Stéphane; Pignet, Patricia; Toffin, Laurent

    2011-01-01

    Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the “active” archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano. PMID:21335391

  18. Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

    Directory of Open Access Journals (Sweden)

    Yu Peng

    Full Text Available Box C/D ribonucleoprotein particles (RNPs are 2'-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2'-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.

  19. Bacterial and archaeal community structures in the Arctic deep-sea sediment

    Institute of Scientific and Technical Information of China (English)

    LI Yan; LIU Qun; LI Chaolun; DONG Yi; ZHANG Wenyan; ZHANG Wuchang; XIAO Tian

    2015-01-01

    Microbial community structures in the Arctic deep-sea sedimentary ecosystem are determined by organic matter input, energy availability, and other environmental factors. However, global warming and earlier ice-cover melting are affecting the microbial diversity. To characterize the Arctic deep-sea sediment microbial diversity and its rela-tionship with environmental factors, we applied Roche 454 sequencing of 16S rDNA amplicons from Arctic deep-sea sediment sample. Both bacterial and archaeal communities’ richness, compositions and structures as well as tax-onomic and phylogenetic affiliations of identified clades were characterized. Phylotypes relating to sulfur reduction and chemoorganotrophic lifestyle are major groups in the bacterial groups;while the archaeal community is domi-nated by phylotypes most closely related to the ammonia-oxidizing Thaumarchaeota (96.66%) and methanogenic Euryarchaeota (3.21%). This study describes the microbial diversity in the Arctic deep marine sediment (>3 500 m) near the North Pole and would lay foundation for future functional analysis on microbial metabolic processes and pathways predictions in similar environments.

  20. Phylogeny of bacterial and archaeal genomes using conserved genes: supertrees and supermatrices.

    Directory of Open Access Journals (Sweden)

    Jenna Morgan Lang

    Full Text Available Over 3000 microbial (bacterial and archaeal genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA, as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a "primary concordance" tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.

  1. Archaeal membrane-associated proteases: insights on Haloferax volcanii and other haloarchaea

    Directory of Open Access Journals (Sweden)

    Maria Ines Giménez

    2015-02-01

    Full Text Available The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

  2. Overexpression, purification and crystallization of an archaeal DNA ligase from Pyrococcus furiosus

    International Nuclear Information System (INIS)

    Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 Å resolution. DNA ligases seal single-strand breaks in double-stranded DNA and their function is essential to maintain the integrity of the genome during various aspects of DNA metabolism, such as replication, excision repair and recombination. DNA-strand breaks are frequently generated as reaction intermediates in these events and the sealing of these breaks depends solely on the proper function of DNA ligase. Crystals of the archaeal DNA ligase from Pyrococcus furiosus were obtained using 6.6%(v/v) ethanol as a precipitant and diffracted X-rays to 1.7 Å resolution. They belong to the monoclinic space group P21, with unit-cell parameters a = 61.1, b = 88.3, c = 63.4 Å, β = 108.9°. The asymmetric unit contains one ligase molecule

  3. Ecological structuring of bacterial and archaeal taxa in surface ocean waters.

    Science.gov (United States)

    Yilmaz, Pelin; Iversen, Morten H; Hankeln, Wolfgang; Kottmann, Renzo; Quast, Christian; Glöckner, Frank O

    2012-08-01

    The Global Ocean Sampling (GOS) expedition is currently the largest and geographically most comprehensive metagenomic dataset, including samples from the Atlantic, Pacific, and Indian Oceans. This study makes use of the wide range of environmental conditions and habitats encompassed within the GOS sites in order to investigate the ecological structuring of bacterial and archaeal taxon ranks. Community structures based on taxonomically classified 16S ribosomal RNA (rRNA) gene fragments at phylum, class, order, family, and genus rank levels were examined using multivariate statistical analysis, and the results were inspected in the context of oceanographic environmental variables and structured habitat classifications. At all taxon rank levels, community structures of neritic, oceanic, estuarine biomes, as well as other exotic biomes (salt marsh, lake, mangrove), were readily distinguishable from each other. A strong structuring of the communities with chlorophyll a concentration and a weaker yet significant structuring with temperature and salinity were observed. Furthermore, there were significant correlations between community structures and habitat classification. These results were used for further investigation of one-to-one relationships between taxa and environment and provided indications for ecological preferences shaped by primary production for both cultured and uncultured bacterial and archaeal clades. PMID:22416918

  4. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

    Directory of Open Access Journals (Sweden)

    David S. Shin

    2014-01-01

    Full Text Available As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.

  5. Geographic distribution of archaeal ammonia oxidizing ecotypes in the Atlantic Ocean

    Directory of Open Access Journals (Sweden)

    Eva eSintes

    2016-02-01

    Full Text Available In marine ecosystems, Thaumarchaeota are most likely the major ammonia oxidizers. While ammonia concentrations vary by about two orders of magnitude in the oceanic water column, archaeal ammonia oxidizers (AOA vary by only one order of magnitude from surface to bathypelagic waters. Thus, the question arises whether the key enzyme responsible for ammonia oxidation, ammonia monooxygenase (amo, exhibits different affinities to ammonia along the oceanic water column and consequently, whether there are different ecotypes of AOA present in the oceanic water column. We determined the abundance and phylogeny of archaeal ammonia oxidizers (AOA based on their amoA gene. Two ecotypes of AOA exhibited a distribution pattern reflecting the reported availability of ammonia and the physico-chemical conditions throughout the Atlantic, and from epi- to bathypelagic waters. The distinction between these two ecotypes was not only detectable at the nucleotide level. Consistent changes were also detected at the amino acid level. These changes include substitutions of polar to hydrophobic amino acid, and glycine substitutions that could have an effect on the configuration of the amo protein and thus, on its activity. Although we cannot identify the specific effect, the ratio of non-synonymous to synonymous substitutions (dN/dS between the two ecotypes indicates a strong positive selection between them. Consequently, our results point to a certain degree of environmental selection on these two ecotypes that have led to their niche specialization.

  6. Bacterial and archaeal dynamics in phylogeny and function in the North Atlantic deep waters

    Science.gov (United States)

    Herndl, G. J.; Brink, M.; Agogue, H.

    2009-04-01

    The diversity and specific functional aspects linked to the N cycle of the bacterio- and archaeoplankton were investigated in the major deep water masses of the North Atlantic following the main driver of the thermohaline circulation, the North Atlantic Deep Water, from 65°N to 5°S. The phylogenetic composition of Bacteria and Archaea is not only depth-dependent but, specific water masses harbor specific prokaryotic communities. The specific composition of these communities in a particular water mass is maintained even over large distances. The distribution of archaeal and bacterial amoA genes were also determined. Archaeal amoA copy numbers decreased drastically with depth especially in the eastern subtropical Atlantic. This coincides with the lower nutrient concentration of the deep waters in the southern parts of the North Atlantic and the older age of the deep-water masses there. These data demonstrate that the diversity and potential nitrification activity are closely linked to the hydrology and chemical characteristics of the major water masses in the North Atlantic.

  7. Archaeal Life on Tangkuban Perahu- Sampling and Culture Growth in Indonesian Laboratories

    Directory of Open Access Journals (Sweden)

    SRI HANDAYANI

    2012-09-01

    Full Text Available The aim of the expedition to Tangkuban Perahu, West Java was to obtain archaeal samples from the solfatara fields located in Domas crater. This was one of the places, where scientists from the University of Regensburg Germany had formerly isolated Indonesian archaea, especially Thermoplasma and Sulfolobus species but not fully characterized. We collected five samples from mud holes with temperatures from 57 to 88 oC and pH of 1.5-2. A portion of each sample was grown at the University of Regensburg in modified Allen’s medium at 80 oC. From four out of five samples enrichment cultures were obtained, autotrophically on elemental sulphur and heterotrophically on sulfur and yeast extract; electron micrographs are presented. In the laboratories of Universitas Indonesia the isolates were cultured at 55-60 oC in order to grow tetraetherlipid synthesizing archaea, both Thermoplasmatales and Sulfolobales. Here, we succeeded to culture the same type of archaeal cells, which had been cultured in Regensburg, probably a Sulfolobus species and in Freundt’s medium, Thermoplasma species. The harvested cells are documented by phase contrast microscope equipped with a digital camera. Our next steps will be to further characterize genetically the cultured cells from Tangkuban Perahu isolates.

  8. Multiple Features Based Two-stage Hybrid Classifier Ensembles for Subcellular Phenotype Images Classification

    OpenAIRE

    Bailing Zhang - China; Tuan D. Pham - Australia

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. As an interesting ``bio-image informatics'' application, an automatic, reliable and efficient prediction system for protein subcellular localization can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, we propose a two-stage multiple classifier system to improve classification...

  9. Predicting multiplex subcellular localization of proteins using protein-protein interaction network: a comparative study

    OpenAIRE

    Jiang Jonathan Q; Wu Maoying

    2012-01-01

    Abstract Background Proteins that interact in vivo tend to reside within the same or "adjacent" subcellular compartments. This observation provides opportunities to reveal protein subcellular localization in the context of the protein-protein interaction (PPI) network. However, so far, only a few efforts based on heuristic rules have been made in this regard. Results We systematically and quantitatively validate the hypothesis that proteins physically interacting with each other probably shar...

  10. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    Science.gov (United States)

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  11. Linking Subcellular Disturbance to Physiological Behavior and Toxicity Induced by Quantum Dots in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Qin; Zhou, Yanfeng; Song, Bin; Zhong, Yiling; Wu, Sicong; Cui, Rongrong; Cong, Haixia; Su, Yuanyuan; Zhang, Huimin; He, Yao

    2016-06-01

    The wide-ranging applications of fluorescent semiconductor quantum dots (QDs) have triggered increasing concerns about their biosafety. Most QD-related toxicity studies focus on the subcellular processes in cultured cells or global physiological effects on whole animals. However, it is unclear how QDs affect subcellular processes in living organisms, or how the subcellular disturbance contributes to the overall toxicity. Here the behavior and toxicity of QDs of three different sizes in Caenorhabditis elegans (C. elegans) are systematically investigated at both the systemic and the subcellular level. Specifically, clear size-dependent distribution and toxicity of the QDs in the digestive tract are observed. Short-term exposure of QDs leads to acute toxicity on C. elegans, yet incurring no lasting, irreversible damage. In contrast, chronic exposure of QDs severely inhibits development and shortens lifespan. Subcellular analysis reveals that endocytosis and nutrition storage are disrupted by QDs, which likely accounts for the severe deterioration in growth and longevity. This work reveals that QDs invasion disrupts key subcellular processes in living organisms, and may cause permanent damage to the tissues and organs over long-term retention. The findings provide invaluable information for safety evaluations of QD-based applications and offer new opportunities for design of novel nontoxic nanoprobes. PMID:27121203

  12. Predicting protein subcellular locations using hierarchical ensemble of Bayesian classifiers based on Markov chains

    Directory of Open Access Journals (Sweden)

    Eils Roland

    2006-06-01

    Full Text Available Abstract Background The subcellular location of a protein is closely related to its function. It would be worthwhile to develop a method to predict the subcellular location for a given protein when only the amino acid sequence of the protein is known. Although many efforts have been made to predict subcellular location from sequence information only, there is the need for further research to improve the accuracy of prediction. Results A novel method called HensBC is introduced to predict protein subcellular location. HensBC is a recursive algorithm which constructs a hierarchical ensemble of classifiers. The classifiers used are Bayesian classifiers based on Markov chain models. We tested our method on six various datasets; among them are Gram-negative bacteria dataset, data for discriminating outer membrane proteins and apoptosis proteins dataset. We observed that our method can predict the subcellular location with high accuracy. Another advantage of the proposed method is that it can improve the accuracy of the prediction of some classes with few sequences in training and is therefore useful for datasets with imbalanced distribution of classes. Conclusion This study introduces an algorithm which uses only the primary sequence of a protein to predict its subcellular location. The proposed recursive scheme represents an interesting methodology for learning and combining classifiers. The method is computationally efficient and competitive with the previously reported approaches in terms of prediction accuracies as empirical results indicate. The code for the software is available upon request.

  13. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    Science.gov (United States)

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

  14. Birth of Archaeal Cells: Molecular Phylogenetic Analyses of G1P Dehydrogenase, G3P Dehydrogenases, and Glycerol Kinase Suggest Derived Features of Archaeal Membranes Having G1P Polar Lipids

    Science.gov (United States)

    2016-01-01

    Bacteria and Eukarya have cell membranes with sn-glycerol-3-phosphate (G3P), whereas archaeal membranes contain sn-glycerol-1-phosphate (G1P). Determining the time at which cells with either G3P-lipid membranes or G1P-lipid membranes appeared is important for understanding the early evolution of terrestrial life. To clarify this issue, we reconstructed molecular phylogenetic trees of G1PDH (G1P dehydrogenase; EgsA/AraM) which is responsible for G1P synthesis and G3PDHs (G3P dehydrogenase; GpsA and GlpA/GlpD) and glycerol kinase (GlpK) which is responsible for G3P synthesis. Together with the distribution of these protein-encoding genes among archaeal and bacterial groups, our phylogenetic analyses suggested that GlpA/GlpD in the Commonote (the last universal common ancestor of all extant life with a cellular form, Commonote commonote) acquired EgsA (G1PDH) from the archaeal common ancestor (Commonote archaea) and acquired GpsA and GlpK from a bacterial common ancestor (Commonote bacteria). In our scenario based on this study, the Commonote probably possessed a G3P-lipid membrane synthesized enzymatically, after which the archaeal lineage acquired G1PDH followed by the replacement of a G3P-lipid membrane with a G1P-lipid membrane.

  15. Predicting Protein Subcellular Location Using Digital Signal Processing

    Institute of Scientific and Technical Information of China (English)

    Yu-Xi PAN; Da-Wei LI; Yun DUAN; Zhi-Zhou ZHANG; Ming-Qing XU; Guo-Yin FENG; Lin HE

    2005-01-01

    The biological functions of a protein are closely related to its attributes in a cell. With the rapid accumulation of newly found protein sequence data in databanks, it is highly desirable to develop an automated method for predicting the subcellular location of proteins. The establishment of such a predictor will expedite the functional determination of newly found proteins and the process of prioritizing genes and proteins identified by genomic efforts as potential molecular targets for drug design. The traditional algorithms for predicting these attributes were based solely on amino acid composition in which no sequence order effect was taken into account. To improve the prediction quality, it is necessary to incorporate such an effect. However, the number of possible patterns in protein sequences is extremely large, posing a formidable difficulty for realizing this goal. To deal with such difficulty, a well-developed tool in digital signal processing named digital Fourier transform (DFT) [1] was introduced. After being translated to a digital signal according to the hydrophobicity of each amino acid, a protein was analyzed by DFT within the frequency domain. A set of frequency spectrum parameters, thus obtained, were regarded as the factors to represent the sequence order effect. A significant improvement in prediction quality was observed by incorporating the frequency spectrum parameters with the conventional amino acid composition. One of the crucial merits of this approach is that many existing tools in mathematics and engineering can be easily applied in the predicting process. It is anticipated that digital signal processing may serve as a useful vehicle for many other protein science areas.

  16. A sub-cellular viscoelastic model for cell population mechanics.

    Directory of Open Access Journals (Sweden)

    Yousef Jamali

    Full Text Available Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and 'in silico' (computational models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM, effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the

  17. Subcellular localization and compartmentation of thiamine derivatives in rat brain.

    Science.gov (United States)

    Bettendorff, L; Wins, P; Lesourd, M

    1994-05-26

    The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate. PMID:8186256

  18. Archaeal tetraether membrane lipid fluxes in the northeastern Pacific and the Arabian Sea: implications for TEX86 paleothermometry

    NARCIS (Netherlands)

    Wuchter, C.; Schouten, S.; Wakeham, S.G.; Sinninghe Damsté, J.S.

    2006-01-01

    The newly introduced temperature proxy, the tetraether index of archaeal lipids with 86 carbon atoms (TEX86), is based on the number of cyclopentane moieties in the glycerol dialkyl glycerol tetraether (GDGT) lipids of marine Crenarchaeota. The composition of sedimentary GDGTs used for TEX86 paleoth

  19. Structure and genome organization of AFV2, a novel archaeal lipothrixvirus with unusual terminal and core structures

    DEFF Research Database (Denmark)

    Häring, Monika; Vestergaard, Gisle Alberg; Brügger, Kim;

    2005-01-01

    A novel filamentous virus, AFV2, from the hyperthermophilic archaeal genus Acidianus shows structural similarity to lipothrixviruses but differs from them in its unusual terminal and core structures. The double-stranded DNA genome contains 31,787 bp and carries eight open reading frames homologous...

  20. Structural and genomic properties of the hyperthermophilic archaeal virus ATV with an extracellular stage of the reproductive cycle

    DEFF Research Database (Denmark)

    Prangishvili, David; Vestergaard, Gisle Alberg; Häring, Monika;

    2006-01-01

    A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon-shaped tail-less particles, after which they develop long...

  1. Spatial isolation and environmental factors drive distinct bacterial and archaeal communities in different types of petroleum reservoirs in China

    Science.gov (United States)

    Gao, Peike; Tian, Huimei; Wang, Yansen; Li, Yanshu; Li, Yan; Xie, Jinxia; Zeng, Bing; Zhou, Jiefang; Li, Guoqiang; Ma, Ting

    2016-02-01

    To investigate the spatial distribution of microbial communities and their drivers in petroleum reservoir environments, we performed pyrosequencing of microbial partial 16S rRNA, derived from 20 geographically separated water-flooding reservoirs, and two reservoirs that had not been flooded, in China. The results indicated that distinct underground microbial communities inhabited the different reservoirs. Compared with the bacteria, archaeal alpha-diversity was not strongly correlated with the environmental variables. The variation of the bacterial and archaeal community compositions was affected synthetically, by the mining patterns, spatial isolation, reservoir temperature, salinity and pH of the formation brine. The environmental factors explained 64.22% and 78.26% of the total variance for the bacterial and archaeal communities, respectively. Despite the diverse community compositions, shared populations (48 bacterial and 18 archaeal genera) were found and were dominant in most of the oilfields. Potential indigenous microorganisms, including Carboxydibrachium, Thermosinus, and Neptunomonas, were only detected in a reservoir that had not been flooded with water. This study indicates that: 1) the environmental variation drives distinct microbial communities in different reservoirs; 2) compared with the archaea, the bacterial communities were highly heterogeneous within and among the reservoirs; and 3) despite the community variation, some microorganisms are dominant in multiple petroleum reservoirs.

  2. Detection and analysis of elusive members of a novel and diverse archaeal community within a thermal spring streamer consortium.

    Science.gov (United States)

    Colman, Daniel R; Thomas, Raquela; Maas, Kendra R; Takacs-Vesbach, Cristina D

    2015-03-01

    Recent metagenomic analyses of Yellowstone National Park (YNP) thermal spring communities suggested the presence of minor archaeal populations that simultaneous PCR-based assays using traditional 'universal' 16S rRNA gene primers failed to detect. Here we use metagenomics to identify PCR primers effective at detecting elusive members of the Archaea, assess their efficacy, and describe the diverse and novel archaeal community from a circum-neutral thermal spring from the Bechler region of YNP. We determined that a less commonly used PCR primer, Arch349F, captured more diversity in this spring than the widely used A21F primer. A search of the PCR primers against the RDP 16S rRNA gene database indicated that Arch349F also captured the largest percentage of Archaea, including 41 % more than A21F. Pyrosequencing using the Arch349F primer recovered all of the phylotypes present in the clone-based portion of the study and the metagenome of this spring in addition to several other populations of Archaea, some of which are phylogenetically novel. In contrast to the lack of amplification with traditional 16S rRNA gene primers, our comprehensive analyses suggested a diverse archaeal community in the Bechler spring, with implications for recently discovered groups such as the Geoarchaeota and other undescribed archaeal groups.

  3. Archaeal ammonia oxidation in volcanic grassland soils of Iceland. Effects of elevated temperature and N availability on processes and organisms

    NARCIS (Netherlands)

    Daebeler, A.

    2014-01-01

    Thaumarchaea are recognized today as the most abundant and ubiquitously dis­tributed archaeal organisms, especially in the oceans and soil. Their phylogenetic placement as a phylum, the capability of all cultivated Thaumarchaea to oxidize ammonia for energy conservation as well as many further aspec

  4. Archaeal amoA gene diversity points to distinct biogeography of ammonia-oxidizing Crenarchaeota in the ocean

    NARCIS (Netherlands)

    Sintes, Eva; Bergauer, Kristin; De Corte, Daniele; Yokokawa, Taichi; Herndl, Gerhard J.

    2013-01-01

    Mesophilic ammonia-oxidizing Archaea (AOA) are abundant in a diverse range of marine environments, including the deep ocean, as revealed by the quantification of the archaeal amoA gene encoding the alpha-subunit of the ammonia monooxygenase. Using two different amoA primer sets, two distinct ecotype

  5. Forest strata drive spatial structure of bacterial and archaeal communities and microbial methane cycling in neotropical bromeliad wetlands

    Science.gov (United States)

    Martinson, Guntars; Brandt, Franziska; Conrad, Ralf

    2016-04-01

    Several thousands of tank bromeliads per hectare of neotropical forest create a unique wetland ecosystem that harbors diverse communities of archaea and bacteria and emit substantial amounts of methane. We studied spatial distribution of archaeal and bacterial communities, microbial methane cycling and their environmental drivers in tank bromeliad wetlands. We selected tank bromeliads of different species and functional types (terrestrial and canopy bromeliads) in a neotropical montane forest of Southern Ecuador and sampled the organic tank slurry. Archaeal and bacterial communities were characterized using terminal-restriction fragment length polymorphism (T-RFLP) and Illumina MiSeq sequencing, respectively, and linked with physico-chemical tank-slurry properties. Additionally, we performed tank-slurry incubations to measure methane production potential, stable carbon isotope fractionation and pathway of methane formation. Archaeal and bacterial community composition in bromeliad wetlands was dominated by methanogens and by Alphaproteobacteria, respectively, and did not differ between species but between functional types. Hydrogenotrophic Methanomicrobiales were the dominant methanogens among all bromeliads but the relative abundance of aceticlastic Methanosaetaceae increased in terrestrial bromeliads. Complementary, hydrogenotrophic methanogenesis was the dominant pathway of methane formation but the relative contribution of aceticlastic methanogenesis increased in terrestrial bromeliads and led to a concomitant increase in total methane production. Rhodospirillales were characteristic for canopy bromeliads, Planctomycetales and Actinomycetalis for terrestrial bromeliads. While nitrogen concentration and pH explained 32% of the archaeal community variability, 29% of the bacterial community variability was explained by nitrogen, acetate and propionate concentrations. Our study demonstrates that bromeliad functional types, associated with different forest strata

  6. Archaeal community in a human-disturbed watershed in southeast China: diversity, distribution, and responses to environmental changes.

    Science.gov (United States)

    Hu, Anyi; Wang, Hongjie; Li, Jiangwei; Liu, Jing; Chen, Nengwang; Yu, Chang-Ping

    2016-05-01

    The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of β-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities. PMID:26810199

  7. A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition

    Directory of Open Access Journals (Sweden)

    Hiromi Daiyasu

    2005-01-01

    Full Text Available Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS, from Methanothermobacter thermautotrophicus, has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS, phosphatidylglycerol synthase (PGS and phosphatidylinositol synthase (PIS derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea

  8. Generation of the Brucella melitensis ORFeome Version 1.1

    OpenAIRE

    Dricot, A. (Amélie); Rual, J. J. (Jean François); Lamesch, P; BERTIN, N.; Dupuy, D. (Denis); Hao, T.; Lambert, C; Hallez, R.; Delroisse, J.M. (Jean Marc); Vandenhaute, J; Lopez-Goñi, I. (Ignacio); Moriyon, I. (Ignacio); Garcia-Lobo, J M; Sangari, F.J. (Félix Javier); MacMillan, A.P.

    2004-01-01

    The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and cons...

  9. The σ enigma: bacterial σ factors, archaeal TFB and eukaryotic TFIIB are homologs.

    Science.gov (United States)

    Burton, Samuel P; Burton, Zachary F

    2014-01-01

    Structural comparisons of initiating RNA polymerase complexes and structure-based amino acid sequence alignments of general transcription initiation factors (eukaryotic TFIIB, archaeal TFB and bacterial σ factors) show that these proteins are homologs. TFIIB and TFB each have two-five-helix cyclin-like repeats (CLRs) that include a C-terminal helix-turn-helix (HTH) motif (CLR/HTH domains). Four homologous HTH motifs are present in bacterial σ factors that are relics of CLR/HTH domains. Sequence similarities clarify models for σ factor and TFB/TFIIB evolution and function and suggest models for promoter evolution. Commitment to alternate modes for transcription initiation appears to be a major driver of the divergence of bacteria and archaea. PMID:25483602

  10. Biological Membranes in Extreme Conditions: Simulations of Anionic Archaeal Tetraether Lipid Membranes.

    Directory of Open Access Journals (Sweden)

    Luis Felipe Pineda De Castro

    Full Text Available In contrast to the majority of organisms that have cells bound by di-ester phospholipids, archaeal membranes consist of di- and tetraether phospholipids. Originating from organisms that withstand harsh conditions (e.g., low pH and a wide range of temperatures such membranes have physical properties that make them attractive materials for biological research and biotechnological applications. We developed force-field parameters based on the widely used Generalized Amber Force Field (GAFF to enable the study of anionic tetraether membranes of the model archaean Sulfolobus acidocaldarius by computer simulations. The simulations reveal that the physical properties of these unique membranes depend on the number of cyclopentane rings included in each lipid unit, and on the size of cations that are used to ensure charge neutrality. This suggests that the biophysical properties of Sulfolobus acidocaldarius cells depend not only on the compositions of their membranes but also on the media in which they grow.

  11. Archaeal Abundance across a pH Gradient in an Arable Soil and Its Relationship to Bacterial and Fungal Growth Rates

    OpenAIRE

    Bengtson, Per; Sterngren, Anna E.; Rousk, Johannes

    2012-01-01

    Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abund...

  12. MED: a new non-supervised gene prediction algorithm for bacterial and archaeal genomes

    Directory of Open Access Journals (Sweden)

    Yang Yi-Fan

    2007-03-01

    Full Text Available Abstract Background Despite a remarkable success in the computational prediction of genes in Bacteria and Archaea, a lack of comprehensive understanding of prokaryotic gene structures prevents from further elucidation of differences among genomes. It continues to be interesting to develop new ab initio algorithms which not only accurately predict genes, but also facilitate comparative studies of prokaryotic genomes. Results This paper describes a new prokaryotic genefinding algorithm based on a comprehensive statistical model of protein coding Open Reading Frames (ORFs and Translation Initiation Sites (TISs. The former is based on a linguistic "Entropy Density Profile" (EDP model of coding DNA sequence and the latter comprises several relevant features related to the translation initiation. They are combined to form a so-called Multivariate Entropy Distance (MED algorithm, MED 2.0, that incorporates several strategies in the iterative program. The iterations enable us to develop a non-supervised learning process and to obtain a set of genome-specific parameters for the gene structure, before making the prediction of genes. Conclusion Results of extensive tests show that MED 2.0 achieves a competitive high performance in the gene prediction for both 5' and 3' end matches, compared to the current best prokaryotic gene finders. The advantage of the MED 2.0 is particularly evident for GC-rich genomes and archaeal genomes. Furthermore, the genome-specific parameters given by MED 2.0 match with the current understanding of prokaryotic genomes and may serve as tools for comparative genomic studies. In particular, MED 2.0 is shown to reveal divergent translation initiation mechanisms in archaeal genomes while making a more accurate prediction of TISs compared to the existing gene finders and the current GenBank annotation.

  13. Seasonality and resource availability control bacterial and archaeal communities in soils of a temperate beech forest.

    Science.gov (United States)

    Rasche, Frank; Knapp, Daniela; Kaiser, Christina; Koranda, Marianne; Kitzler, Barbara; Zechmeister-Boltenstern, Sophie; Richter, Andreas; Sessitsch, Angela

    2011-03-01

    It was hypothesized that seasonality and resource availability altered through tree girdling were major determinants of the phylogenetic composition of the archaeal and bacterial community in a temperate beech forest soil. During a 2-year field experiment, involving girdling of beech trees to intercept the transfer of easily available carbon (C) from the canopy to roots, members of the dominant phylogenetic microbial phyla residing in top soils under girdled versus untreated control trees were monitored at bimonthly intervals through 16S rRNA gene-based terminal restriction fragment length polymorphism profiling and quantitative PCR analysis. Effects on nitrifying and denitrifying groups were assessed by measuring the abundances of nirS and nosZ genes as well as bacterial and archaeal amoA genes. Seasonal dynamics displayed by key phylogenetic and nitrogen (N) cycling functional groups were found to be tightly coupled with seasonal alterations in labile C and N pools as well as with variation in soil temperature and soil moisture. In particular, archaea and acidobacteria were highly responsive to soil nutritional and soil climatic changes associated with seasonality, indicating their high metabolic versatility and capability to adapt to environmental changes. For these phyla, significant interrelations with soil chemical and microbial process data were found suggesting their potential, but poorly described contribution to nitrification or denitrification in temperate forest soils. In conclusion, our extensive approach allowed us to get novel insights into effects of seasonality and resource availability on the microbial community, in particular on hitherto poorly studied bacterial phyla and functional groups.

  14. SIMS ion microscopy as a novel, practical tool for subcellular chemical imaging in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, S

    2003-01-15

    The development of cryogenic sample preparations, subcellular image quantification schemes, and correlative confocal laser scanning microscopy and ion microscopy have made dynamic SIMS a versatile tool in biology and medicine. For example, ion microscopy can provide much needed, novel information on calcium influx and intracellular calcium stores at organelle resolution in normal and transformed cells in order to better understand the altered calcium signaling in malignant cells. 3-D SIMS imaging of cells revealed dynamic gradients of calcium in cells undergoing mitosis and cytokinesis. Studies of subcellular localization of anticancer drugs is another area of research where ion microscopy can provide novel observations in many types of cancers. Ion microscopy is already an essential tool in boron neutron capture therapy (BNCT) of brain cancer as it can be used to quantitatively image the subcellular location of boron in cells and tissues. This information is critically needed for testing the efficacy of boronated agents and for calculations of radiation dosimetry.

  15. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    Science.gov (United States)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  16. Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

    Science.gov (United States)

    Perrière, Guy; Thioulouse, Jean

    2003-02-01

    Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

  17. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier.

    Science.gov (United States)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  18. MOLECULAR CLONING, EXPRESSION AND SUBCELLULAR LOCALIZATION OF HUMAN OCT-4 PROTEIN

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To clone, express, purify human Oct-4 and detect its subcellular localization.Methods Human Oct-4 cDNA was amplified by RT-PCR strategy. Oct-4 protein was induced and expressed in BL21 (DE3) strain. Furthermore, the protein was purified with Ni-NTA resin. Subsequently, site-directed mutagenesis of Oct-4 (aa 236 -240) was introduced. Finally, the subcellular localization of wide type Oct-4 and mutant Oct-4 was examined by immunofluorescent cytochemistry staining and confocal laser scanning microscope analysis.Results The full length cDNA of human Oct-4 was 1083bp. Human Oct-4 encoded a 55 kd protein by prokaryotic vector in E coli. Compared with pure nuclear localization of wide type Oct-4, mutant Oct-4 was mostly enriched in the cytoplasm. Conclusion The cloning, expression and investigation of subcellular localization of human Oct-4 are basis of studying its biological function.

  19. Multi-label multi-kernel transfer learning for human protein subcellular localization.

    Directory of Open Access Journals (Sweden)

    Suyu Mei

    Full Text Available Recent years have witnessed much progress in computational modelling for protein subcellular localization. However, the existing sequence-based predictive models demonstrate moderate or unsatisfactory performance, and the gene ontology (GO based models may take the risk of performance overestimation for novel proteins. Furthermore, many human proteins have multiple subcellular locations, which renders the computational modelling more complicated. Up to the present, there are far few researches specialized for predicting the subcellular localization of human proteins that may reside in multiple cellular compartments. In this paper, we propose a multi-label multi-kernel transfer learning model for human protein subcellular localization (MLMK-TLM. MLMK-TLM proposes a multi-label confusion matrix, formally formulates three multi-labelling performance measures and adapts one-against-all multi-class probabilistic outputs to multi-label learning scenario, based on which to further extends our published work GO-TLM (gene ontology based transfer learning model for protein subcellular localization and MK-TLM (multi-kernel transfer learning based on Chou's PseAAC formulation for protein submitochondria localization for multiplex human protein subcellular localization. With the advantages of proper homolog knowledge transfer, comprehensive survey of model performance for novel protein and multi-labelling capability, MLMK-TLM will gain more practical applicability. The experiments on human protein benchmark dataset show that MLMK-TLM significantly outperforms the baseline model and demonstrates good multi-labelling ability for novel human proteins. Some findings (predictions are validated by the latest Swiss-Prot database. The software can be freely downloaded at http://soft.synu.edu.cn/upload/msy.rar.

  20. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    Directory of Open Access Journals (Sweden)

    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  1. Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Beaumelle, Léa [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Gimbert, Frédéric [Laboratoire Chrono-Environnement, UMR 6249 University of Franche-Comté/CNRS Usc INRA, 16 route de Gray, 25030 Besançon Cedex (France); Hedde, Mickaël [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Guérin, Annie [INRA, US 0010 LAS Laboratoire d' analyses des sols, 273 rue de Cambrai, 62000 Arras (France); Lamy, Isabelle, E-mail: lamy@versailles.inra.fr [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France)

    2015-07-01

    Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl{sub 2}-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl{sub 2} extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm. - Highlights: • Earthworms were exposed to a wide panel of historically contaminated soils • Subcellular partitioning of Cd, Pb and Zn was investigated in earthworms • Three proxies of soil metal availability were

  2. Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

    Science.gov (United States)

    Datta, R K; Antopol, W; Ghosh, J J

    1977-01-01

    Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

  3. Archaeal and bacterial tetraether lipids in tropical ponds with contrasted salinity (Guadeloupe, French West Indies): Implications for tetraether-based environmental proxies

    OpenAIRE

    Huguet, Arnaud; Grossi, Vincent; Belmahdi, Imène; Fosse, Céline; Derenne, Sylvie

    2015-01-01

    International audience The occurrence and distribution of archaeal and bacterial glycerol dialkyl glycerol tetraether lipids (GDGTs) in continental saline environments have been rarely investigated. Here, the abundance and distribution of archaeal isoprenoid GDGTs (iGDGTs) and archaeol, and of bacterial branched GDGTs (brGDGTs) in four tropical water ponds of contrasting salinity in two islands from the French Western Indies, Grande-Terre and La Désirade, have been determined. The sediment...

  4. Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

    OpenAIRE

    Frade, Pedro R.; Katharina Roll; Kristin Bergauer; Herndl, Gerhard J.

    2016-01-01

    Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and ...

  5. Responses of bacterial and archaeal communities to nitrate stimulation after oil pollution in mangrove sediment revealed by Illumina sequencing.

    Science.gov (United States)

    Wang, Lei; Huang, Xu; Zheng, Tian-Ling

    2016-08-15

    This study aimed to investigate microbial responses to nitrate stimulation in oiled mangrove mesocosm. Both supplementary oil and nitrate changed the water and sediment chemical properties contributing to the shift of microbial communities. Denitrifying genes nirS and nirK were increased several times by the interaction of oil spiking and nitrate addition. Bacterial chao1 was reduced by oil spiking and further by nitrate stimulation, whereas archaeal chao1 was only inhibited by oil pollution on early time. Sampling depth explained most of variation and significantly impacted bacterial and archaeal communities, while oil pollution only significantly impacted bacterial communities (pexplaining less variation, nitrate addition coupled with oil spiking enhanced the growth of hydrocarbon degraders in mangrove. The findings demonstrate the impacts of environmental factors and their interactions in shaping microbial communities during nitrate stimulation. Our study suggests introducing genera Desulfotignum and Marinobacter into oiled mangrove for bioaugmentation. PMID:27262497

  6. Responses of bacterial and archaeal communities to nitrate stimulation after oil pollution in mangrove sediment revealed by Illumina sequencing.

    Science.gov (United States)

    Wang, Lei; Huang, Xu; Zheng, Tian-Ling

    2016-08-15

    This study aimed to investigate microbial responses to nitrate stimulation in oiled mangrove mesocosm. Both supplementary oil and nitrate changed the water and sediment chemical properties contributing to the shift of microbial communities. Denitrifying genes nirS and nirK were increased several times by the interaction of oil spiking and nitrate addition. Bacterial chao1 was reduced by oil spiking and further by nitrate stimulation, whereas archaeal chao1 was only inhibited by oil pollution on early time. Sampling depth explained most of variation and significantly impacted bacterial and archaeal communities, while oil pollution only significantly impacted bacterial communities (poil spiking enhanced the growth of hydrocarbon degraders in mangrove. The findings demonstrate the impacts of environmental factors and their interactions in shaping microbial communities during nitrate stimulation. Our study suggests introducing genera Desulfotignum and Marinobacter into oiled mangrove for bioaugmentation.

  7. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  8. Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts

    Science.gov (United States)

    Woese, C. R.; Achenbach, L.; Rouviere, P.; Mandelco, L.

    1991-01-01

    A major and too little recognized source of artifact in phylogenetic analysis of molecular sequence data is compositional difference among sequences. The problem becomes particularly acute when alignments contain ribosomal RNAs from both mesophilic and thermophilic species. Among prokaryotes the latter are considerably higher in G + C content than the former, which often results in artificial clustering of thermophilic lineages and their being placed artificially deep in phylogenetic trees. In this communication we review archaeal phylogeny in the light of this consideration, focusing in particular on the phylogenetic position of the sulfate reducing species Archaeoglobus fulgidus, using both 16S rRNA and 23S rRNA sequences. The analysis shows clearly that the previously reported deep branching of the A. fulgidus lineage (very near the base of the euryarchaeal side of the archaeal tree) is incorrect, and that the lineage actually groups with a previously recognized unit that comprises the Methanomicrobiales and extreme halophiles.

  9. Subcellular location of the enzymes of purine breakdown in the yeast Candida famata grown on uric acid

    NARCIS (Netherlands)

    Large, Peter J.; Waterham, Hans R.; Veenhuis, Marten

    1990-01-01

    The subcellular location of the enzymes of purine breakdown in the yeast Candida famata, which grows on uric acid as sole carbon and nitrogen source, has been examined by subcellular fractionation methods. Uricase was confirmed as being peroxisomal, but the other three enzymes, allantoinase, allanto

  10. Non-extremophilic 'extremophiles' - Archaeal dominance in the subsurface and their implication for life

    Science.gov (United States)

    Reitschuler, Christoph; Lins, Philipp; Illmer, Paul

    2014-05-01

    Archaea - besides bacteria and eukaryota constituting the third big domain of life - were so far regarded as typical inhabitants of extreme environments, as indicated by the name (Archaeon, Greek: 'original', 'primal'). Previous research and cultivation successes were basically carried out in habitats characterized by extreme temperature, pH and salinity regimes. Such extreme conditions, as expected at the beginning of the Earth's evolution, are occasionally also prevalent on extraterrestrial planets and moons and make the Archaeal domain a key group to be studied concerning life's evolution and the most likely pioneer organisms to colonize environments that are regarded as hostile. However, in recent years it became obvious that Archaea, in particular non-extremophilic species, can be found almost ubiquitously in marine, freshwater, terrestrial and also subsurface habitats and occasionally outnumber other microbial domains and hold key positions in globally relevant energy and nutrient cycles. Besides extreme environments - the big question remains how to define a parameter as extreme - subsurface and cave environments present a window to the past, where adaptions to early life's conditions can be studied and how microbiomes may be structured in a habitat that represents a refugium on extraterrestrial celestial bodies, were surface conditions might be at first sight too extreme for life. The lower part of the alpine Hundsalm cave in Tyrol (Austria) offered a unique opportunity to study an almost pristine cave habitat, which is separated from the touristic part of the ice cave. The main focus of our research was laid on the microbial communities that were supposed to be in connection with secondary carbonate precipitations ('moonmilk'). For the ascertainment of these so far poorly evaluated structures a multiple approach assessment was chosen to generate a virtually complete picture of these subsurface microbiomes. Thereby, a combination of different cultivation

  11. Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

    Directory of Open Access Journals (Sweden)

    Céline Brochier-Armanet

    2006-01-01

    Full Text Available Reverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. Previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (LGT to the ancestors of the two bacterial hyperthermophilic phyla, Thermotogales and Aquificales. Here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. We found genes coding for reverse gyrase in the genomes of several thermophilic bacteria that belong to phyla other than Aquificales and Thermotogales. Several of these bacteria are not, strictly speaking, hyperthermophiles because their reported optimal growth temperatures are below 80 °C. Furthermore, we detected a reverse gyrase gene in the sequence of the large plasmid of Thermus thermophilus strain HB8, suggesting a possible mechanism of transfer to the T. thermophilus strain HB8 involving plasmids and transposases. The archaeal part of the reverse gyrase tree is congruent with recent phylogenies of the archaeal domain based on ribosomal proteins or RNA polymerase subunits. Although poorly resolved, the complete reverse gyrase phylogeny suggests an ancient acquisition of the gene by bacteria via one or two LGT events, followed by its secondary distribution by LGT within bacteria. Finally, several genes of archaeal origin located in proximity to the reverse gyrase gene in bacterial genomes have bacterial homologues mostly in thermophiles or hyperthermophiles, raising the possibility that they were co-transferred with the reverse gyrase gene. Our new analysis of the reverse gyrase history strengthens the hypothesis that the acquisition of reverse gyrase may have been a crucial evolutionary step in the adaptation of bacteria to high-temperature environments. However, it also questions the role of this enzyme in thermophilic bacteria and the selective advantage its presence could provide.

  12. Archaeal tetraether membrane lipid fluxes in the northeastern Pacific and the Arabian Sea: implications for TEX86 paleothermometry

    OpenAIRE

    Wuchter, C.; Schouten, S.; Wakeham, S.G.; Sinninghe Damsté, J.S.

    2006-01-01

    The newly introduced temperature proxy, the tetraether index of archaeal lipids with 86 carbon atoms (TEX86), is based on the number of cyclopentane moieties in the glycerol dialkyl glycerol tetraether (GDGT) lipids of marine Crenarchaeota. The composition of sedimentary GDGTs used for TEX86 paleothermometry is thought to reflect sea surface temperature (SST). However, marine Crenarchaeota occur ubiquitously in the world oceans over the entire depth range and not just in surface waters. We an...

  13. [Effects of long-term fertilization on bacterial and archaeal diversity and community structure within subtropical red paddy soils].

    Science.gov (United States)

    Yuan, Hong-zhao; Wu, Hao; Ge, Ti-da; Li, Ke-lin; Wu, Jin-shui; Wang, Jiu-rong

    2015-06-01

    Paddy soils not only function as an important sink for "missing carbon" but also play an important role in the production of greenhouse gases such as N2O and CH4. Dynamic changes in greenhouse gases in the atmosphere are closely related to microbially mediated carbon and nitrogen transformation processes occurring in soil. Using soil samples collected from a long-term fertilization experimental site in Taojiang County, subtropical China (established in 1986), we determined the effects of long-term (>25 years) non-fertilization (CK), chemical fertilization (NPK), and NPK combined with rice straw residues (NPKS) on soil bacterial and archaeal community structures. The 16S rRNA genotypes from the three differently treated soils were divided into 9 bacterial phylotypes, mainly including Proteobacteria, Acidobacteria, Chloroflexi, and archaea of Crenarchaeota and Euryarchaeota. The relative abundance of Proteobacteria, Acidobacteria and Crenarchaeota increased in the soils under NPK and NPKS treatments, with the increase being greater in the latter treatment. LUBSHUFF statistical analyses also demonstrated that there was significant difference among the microbial community compositions in CK-, NPK- and NPKS-treated soils. The abundance of bacterial and archaeal 16S rRNA genes ranged from 0.58 x 10(10) to 1.06 x 10(10) copies · g(-1) dry soil and from 1.16 x 10(6) to 1.72 x 10(6) copies · g(-1) dry soil, respectively. Application of fertilizers increased the bacterial and archaeal abundance and diversity in the treated soils, with NPKS > NPK. Long-term chemical and organic applications significantly affected the abundance, diversity and composition of bacterial and archaeal communities in paddy ecosystems. PMID:26572036

  14. Archaeal and bacterial community dynamics and bioprocess performance of a bench-scale two-stage anaerobic digester.

    Science.gov (United States)

    Gonzalez-Martinez, Alejandro; Garcia-Ruiz, Maria Jesus; Rodriguez-Sanchez, Alejandro; Osorio, Francisco; Gonzalez-Lopez, Jesus

    2016-07-01

    Two-stage technologies have been developed for anaerobic digestion of waste-activated sludge. In this study, the archaeal and bacterial community structure dynamics and bioprocess performance of a bench-scale two-stage anaerobic digester treating urban sewage sludge have been studied by the means of high-throughput sequencing techniques and physicochemical parameters such as pH, dried sludge, volatile dried sludge, acid concentration, alkalinity, and biogas generation. The coupled analyses of archaeal and bacterial communities and physicochemical parameters showed a direct relationship between archaeal and bacterial populations and bioprocess performance during start-up and working operation of a two-stage anaerobic digester. Moreover, results demonstrated that archaeal and bacterial community structure was affected by changes in the acid/alkalinity ratio in the bioprocess. Thus, a predominance of the acetoclastic methanogen Methanosaeta was observed in the methanogenic bioreactor at high-value acid/alkaline ratio, while a predominance of Methanomassilicoccaeceae archaea and Methanoculleus genus was observed in the methanogenic bioreactor at low-value acid/alkaline ratio. Biodiversity tag-iTag sequencing studies showed that methanogenic archaea can be also detected in the acidogenic bioreactor, although its biological activity was decreased after 4 months of operation as supported by physicochemical analyses. Also, studies of the VFA producers and VFA consumers microbial populations showed as these microbiota were directly affected by the physicochemical parameters generated in the bioreactors. We suggest that the results obtained in our study could be useful for future implementations of two-stage anaerobic digestion processes at both bench- and full-scale. PMID:26940050

  15. Identification and genomic analysis of transcription factors in archaeal genomes exemplifies their functional architecture and evolutionary origin.

    Science.gov (United States)

    Pérez-Rueda, Ernesto; Janga, Sarath Chandra

    2010-06-01

    Archaea, which represent a large fraction of the phylogenetic diversity of organisms, are prokaryotes with eukaryote-like basal transcriptional machinery. This organization makes the study of their DNA-binding transcription factors (TFs) and their transcriptional regulatory networks particularly interesting. In addition, there are limited experimental data regarding their TFs. In this work, 3,918 TFs were identified and exhaustively analyzed in 52 archaeal genomes. TFs represented less than 5% of the gene products in all the studied species comparable with the number of TFs identified in parasites or intracellular pathogenic bacteria, suggesting a deficit in this class of proteins. A total of 75 families were identified, of which HTH_3, AsnC, TrmB, and ArsR families were universally and abundantly identified in all the archaeal genomes. We found that archaeal TFs are significantly small compared with other protein-coding genes in archaea as well as bacterial TFs, suggesting that a large fraction of these small-sized TFs could supply the probable deficit of TFs in archaea, by possibly forming different combinations of monomers similar to that observed in eukaryotic transcriptional machinery. Our results show that although the DNA-binding domains of archaeal TFs are similar to bacteria, there is an underrepresentation of ligand-binding domains in smaller TFs, which suggests that protein-protein interactions may act as mediators of regulatory feedback, indicating a chimera of bacterial and eukaryotic TFs' functionality. The analysis presented here contributes to the understanding of the details of transcriptional apparatus in archaea and provides a framework for the analysis of regulatory networks in these organisms.

  16. Similarities and Contrasts in the Archaeal Community of Two Japanese Mountains: Mt. Norikura Compared to Mt. Fuji.

    Science.gov (United States)

    Singh, Dharmesh; Takahashi, Koichi; Park, Jungok; Adams, Jonathan M

    2016-02-01

    The community ecology, abundance, and diversity patterns of soil archaea are poorly understood-despite the fact that they are a major branch of life that is ubiquitous and important in nitrogen cycling in terrestrial ecosystems. We set out to investigate the elevational patterns of archaeal ecology, and how these compare with other groups of organisms. Many studies of different groups of organisms (plants, birds, etc.) have shown a series of distinct communities with elevation, and often a diversity maximum in mid-elevations. We investigated the soil archaeal communities on Mt. Norikura, Japan, using 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene. There was a strong mid-elevation maximum in diversity, and a mid-elevation maximum in abundance of soil archaea 16S rRNA and amoA genes. These diversity and abundance maximums could not be correlated with any identifiable soil parameter, nor plant diversity. Discrete, predictable communities of archaea occurred at each elevational level, also not explicable in terms of pH or major nutrients. When we compared the archaeal community and diversity patterns with those found in an earlier study of Mt Fuji, both mountains showed mid-elevation maximums in diversity and abundance of archaea, possibly a result of some common environmental factor such as soil disturbance frequency. However, they showed distinct sets of archaeal communities at similar elevational sampling points. Presumably, the difference reflects their distinct geology (Norikura being andesitic, while Fuji is basaltic) and the resulting combinations of soil chemistry and environmental conditions, although no explanatory variable was found. Clearly, many soil archaea have strongly defined niches and will only occur in a narrow subset of the range of possible climate and soil conditions. The findings of a mid-elevation diversity maximum on Norikura provides a further instance of how widespread this unexplained pattern is in nature, in a wide variety of

  17. Seasonal Changes of Freshwater Ammonia-Oxidizing Archaeal Assemblages and Nitrogen Species in Oligotrophic Alpine Lakes▿ †

    OpenAIRE

    Auguet, Jean-Christophe; Nomokonova, Natalya; Camarero, Lluis; Casamayor, Emilio O.

    2011-01-01

    The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal ...

  18. Quantitative and phylogenetic study of the Deep Sea Archaeal Group in sediments of the arctic mid-ocean spreading ridge

    Directory of Open Access Journals (Sweden)

    Steffen Leth eJørgensen

    2013-10-01

    Full Text Available In marine sediments archaea often constitute a considerable part of the microbial community, of which the Deep Sea Archaeal Group (DSAG is one of the most predominant. Despite their high abundance no members from this archaeal group have so far been characterized and thus their metabolism is unknown. Here we show that the relative abundance of DSAG marker genes can be correlated with geochemical parameters, allowing prediction of both the potential electron donors and acceptors of these organisms. We estimated the abundance of 16S rRNA genes from Archaea, Bacteria and DSAG in 52 sediment horizons from two cores collected at the slow-spreading Arctic Mid-Ocean Ridge, using qPCR. The results indicate that members of the DSAG make up the entire archaeal population in certain horizons and constitute up to ~ 50% of the total microbial community. The quantitative data were correlated to 30 different geophysical and geochemical parameters obtained from the same sediment horizons. We observed a significant correlation between the relative abundance of DSAG 16S rRNA genes and the content of organic carbon (p < 0.0001. Further, significant co-variation with iron oxide, and dissolved iron and manganese (all p < 0.0000, indicated a direct or indirect link to iron and manganese cycling. Neither of these parameters correlated with the relative abundance of archaeal or bacterial 16S rRNA genes, nor did any other major electron donor or acceptor measured. Phylogenetic analysis of DSAG 16S rRNA gene sequences reveals three monophyletic lineages with no apparent habitat-specific distribution. In this study we support the hypothesis that members of the DSAG are tightly linked to the content of organic carbon and directly or indirectly involved in the cycling of iron and/or manganese compounds. Further, we provide a molecular tool to assess their abundance in environmental samples and enrichment cultures.

  19. Insights into archaeal evolution and symbiosis from the genomes of a Nanoarchaeon and its crenarchaeal host from Yellowstone National Park

    Energy Technology Data Exchange (ETDEWEB)

    Podar, Mircea [ORNL; Graham, David E [ORNL; Reysenbach, Anna-Louise [Portland State University; Koonin, Eugene [National Center for Biotechnology Information; Wolf, Yuri [National Center for Biotechnology Information; Makarova, Kira S. [National Center for Biotechnology Information

    2013-01-01

    A hyperthemophilic member of the Nanoarchaeota from Obsidian Pool, a thermal feature in Yellowstone National Park was characterized using single cell isolation and sequencing, together with its putative host, a Sulfolobales archaeon. This first representative of a non-marine Nanoarchaeota (Nst1) resembles Nanoarchaeum equitans by lacking most biosynthetic capabilities, the two forming a deep-branching archaeal lineage. However, the Nst1 genome is over 20% larger, encodes a complete gluconeogenesis pathway and a full complement of archaeal flagellum proteins. Comparison of the two genomes suggests that the marine and terrestrial Nanoarchaeota lineages share a common ancestor that was already a symbiont of another archaeon. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales. The two distinct Nanoarchaeota-host genomic data sets offer insights into the evolution of archaeal symbiosis and parasitism and will further enable studies of the cellular and molecular mechanisms of these relationships.

  20. Phylogenetic analysis of the archaeal community in an alkaline-saline soil of the former lake Texcoco (Mexico).

    Science.gov (United States)

    Valenzuela-Encinas, César; Neria-González, Isabel; Alcántara-Hernández, Rocio J; Enríquez-Aragón, J Arturo; Estrada-Alvarado, Isabel; Hernández-Rodríguez, César; Dendooven, Luc; Marsch, Rodolfo

    2008-03-01

    The soil of the former lake Texcoco is an extreme environment localized in the valley of Mexico City, Mexico. It is highly saline and alkaline, where Na+, Cl(-), HCO3(-) and CO3(2-) are the predominant ions, with a pH ranging from 9.8 to 11.7 and electrolytic conductivities in saturation extracts from 22 to 150 dS m(-1). Metagenomic DNA from the archaeal community was extracted directly from soil and used as template to amplify 16S ribosomal gene by PCR. PCR products were used to construct gene libraries. The ribosomal library showed that the archaeal diversity included Natronococcus sp., Natronolimnobius sp., Natronobacterium sp., Natrinema sp., Natronomonas sp., Halovivax sp., "Halalkalicoccus jeotgali" and novel clades within the family of Halobacteriaceae. Four clones could not be classified. It was found that the archaeal diversity in an alkaline-saline soil of the former lake Texcoco, Mexico, was low, but showed yet uncharacterized and unclassified species.

  1. Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities.

    Science.gov (United States)

    Kittelmann, Sandra; Seedorf, Henning; Walters, William A; Clemente, Jose C; Knight, Rob; Gordon, Jeffrey I; Janssen, Peter H

    2013-01-01

    Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

  2. Phylogenetic and functional analysis of metagenome sequence from high-temperature archaeal habitats demonstrate linkages between metabolic potential and geochemistry

    Directory of Open Access Journals (Sweden)

    William P. Inskeep

    2013-05-01

    Full Text Available Geothermal habitats in Yellowstone National Park (YNP provide an unparalled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (~40-45 Mbase Sanger sequencing per site was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G+C content and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH. These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high temperature systems of YNP.

  3. Archaeal diversity and abundance within different layers of summer sea-ice and seawater from Prydz Bay, Antarctica

    Institute of Scientific and Technical Information of China (English)

    MA Jifei; DU Zongjun; LUO Wei; YU Yong; ZENG Yixin; CHEN Bo; LI Huirong

    2014-01-01

    Fluorescence in-situ hybridization (FISH) and 16S rRNA gene clone library analyses were used to determine the abundance and diversity of archaea in Prydz Bay, Antarctica. Correlation analysis was also performed to assess links between physicochemical parameters and archaeal abundance and diversity within the sea-ice. Samples of sea-ice and seawater were collected during the 26th Chinese National Antarctic Research Expedition. The results of FISH showed that archaea were relatively abundant within the top layer of the sea-ice, and correlation analysis suggested that the concentration of 4NH+ might be one of the main factors underlying this distribution pattern. However, using 16S rRNA gene libraries, archaea were not detected in the top and middle layers of the sea-ice. All archaeal clones obtained from the bottom layer of the sea-ice were grouped into the Marine Group I Crenarchaeota while the archaeal clones from seawater were assigned to Marine Group I Crenarchaeota, Marine Group II Euryarchaeota, and Marine Group III Euryarchaeota. Overall, the ifndings of this study showed that the diversity of archaea in the sea-ice in Prydz Bay was low.

  4. Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

    Directory of Open Access Journals (Sweden)

    Tim Soderberg

    2005-01-01

    Full Text Available A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP, the ribulose monophosphate (RuMP pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium produce ribose-5-phosphate via the nonoxidative PPP (NOPPP, whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1 lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP, the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii probably does not synthesize aromatic amino acids at all.

  5. Phylogenetic and Functional Analysis of Metagenome Sequence from High-Temperature Archaeal Habitats Demonstrate Linkages between Metabolic Potential and Geochemistry.

    Science.gov (United States)

    Inskeep, William P; Jay, Zackary J; Herrgard, Markus J; Kozubal, Mark A; Rusch, Douglas B; Tringe, Susannah G; Macur, Richard E; Jennings, Ryan deM; Boyd, Eric S; Spear, John R; Roberto, Francisco F

    2013-01-01

    Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40-45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and/or microbial mats and subjected to numerous phylogenetic and functional analyses. Analysis of individual sequences (e.g., MEGAN and G + C content) and assemblies from each habitat type revealed the presence of dominant archaeal populations in all environments, 10 of whose genomes were largely reconstructed from the sequence data. Analysis of protein family occurrence, particularly of those involved in energy conservation, electron transport, and autotrophic metabolism, revealed significant differences in metabolic strategies across sites consistent with differences in major geochemical attributes (e.g., sulfide, oxygen, pH). These observations provide an ecological basis for understanding the distribution of indigenous archaeal lineages across high-temperature systems of YNP.

  6. Divergent responses of methanogenic archaeal communities in two rice cultivars to elevated ground-level O3.

    Science.gov (United States)

    Zhang, Jianwei; Tang, Haoye; Zhu, Jianguo; Lin, Xiangui; Feng, Youzhi

    2016-06-01

    Inhibitive effect of elevated ground-level ozone (O3) on paddy methane (CH4) emission varies with rice cultivars. However, little information is available on its microbial mechanism. For this purpose, the responses of methane-metabolizing microorganisms, methanogenic archaea and methanotrophic bacteria to O3 pollution were investigated in the O3-tolerant (YD6) and the O3-sensitive (IIY084) cultivars at two rice growth stages in Free Air Concentration Elevation of O3 (O3-FACE) system of China. It was found that O3 pollution didn't change the abundances of Type I and Type II methanotrophic bacteria at two rice stages. For methanogenic archaea, their abundances in both cultivars were decreased by O3 pollution at the tillering stage. Furthermore, a greater negative influence on methanogenic archaeal community was observed on IIY084 than on YD6: at tillering stage, the alpha diversity indices of methanogenic archaeal community in IIY084 was decreased to a greater extent than in YD6; IIY084 shifted methanogenic archaeal community composition and decreased the abundances and the diversities of Methanosarcinaceae and Methanosaetaceae as well as the abundance of Methanomicrobiales, while the diversity of Methanocellaceae were increased in YD6. These findings indicate that the variations in the responses of paddy CH4 emission to O3 pollution between cultivars could result from the divergent responses of their methanogenic archaea. PMID:26895536

  7. Toxicity and subcellular distribution of cadmium in wheat as affected by dissolved organic acids

    Institute of Scientific and Technical Information of China (English)

    Dandan Li; Dongmei Zhou

    2012-01-01

    We aim to investigate the effects of humic acid (HA) and citric acid (CA) on the toxicity and subcellular distribution of Cd in wheat.Results show that the toxicity and uptake of Cd decreased with increasing HA.The EC50 values of Cd increased from 3.36 μmol/L to 4.96 and 7.33 μmol/L at 50 and 250 mg/L HA,respectively,but decreased to 1.39 μmol/L in the presence of CA based on free ion activity model (FIAM).HA decreased the relative subcellular distribution of Cd in the heat-denatured proteins (decreased from 54% to 33%) but increased Cd in the heat-stable proteins in root (from 25% to 50%) at 7.61 μmol/L {Cd2+} (free Cd activity),which resulted in decreasing Cd toxicity.However,CA increased Cd toxicity due to the increased internalization of Cd although the relative subcellular distributions of Cd exhibited a decrease in the heat-denatured proteins and increase in the granule fraction compared to the control at high-level Cd.The FIAM could not predict the toxicity of Cd in the presence of organic acids.Alternatively,the internal Cd accumulation and subcellular Cd concentration were better to describe the toxicity of Cd to wheat.

  8. Development of a Charged Particle Microbeam for Targeted and Single Particle Subcellular Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yanch, Jacquelyn C.

    2004-03-12

    The development of a charged particle microbeam for single particle, subcellular irradiations at the Massachusetts Institute of Technology Laboratory for Accelerator Beam Applications (MIT LABA) was initiated under this NEER aeard. The Microbeam apparatus makes use of a pre-existing electrostatic accelerator with a horizontal beam tube.

  9. Different subcellular locations of secretome components of Gram-positive bacteria

    NARCIS (Netherlands)

    Buist, Girbe; Ridder, Anja N. J. A.; Kok, Jan; Kuipers, Oscar P.

    2006-01-01

    Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various

  10. Optically-controlled platforms for transfection and single- and sub-cellular surgery

    DEFF Research Database (Denmark)

    Villangca, Mark Jayson; Casey, Duncan; Glückstad, Jesper

    2015-01-01

    Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredic...

  11. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction

    Directory of Open Access Journals (Sweden)

    Kohlbacher Oliver

    2009-09-01

    Full Text Available Abstract Background Knowledge of subcellular localization of proteins is crucial to proteomics, drug target discovery and systems biology since localization and biological function are highly correlated. In recent years, numerous computational prediction methods have been developed. Nevertheless, there is still a need for prediction methods that show more robustness and higher accuracy. Results We extended our previous MultiLoc predictor by incorporating phylogenetic profiles and Gene Ontology terms. Two different datasets were used for training the system, resulting in two versions of this high-accuracy prediction method. One version is specialized for globular proteins and predicts up to five localizations, whereas a second version covers all eleven main eukaryotic subcellular localizations. In a benchmark study with five localizations, MultiLoc2 performs considerably better than other methods for animal and plant proteins and comparably for fungal proteins. Furthermore, MultiLoc2 performs clearly better when using a second dataset that extends the benchmark study to all eleven main eukaryotic subcellular localizations. Conclusion MultiLoc2 is an extensive high-performance subcellular protein localization prediction system. By incorporating phylogenetic profiles and Gene Ontology terms MultiLoc2 yields higher accuracies compared to its previous version. Moreover, it outperforms other prediction systems in two benchmarks studies. MultiLoc2 is available as user-friendly and free web-service, available at: http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc2.

  12. Substrates with patterned extracellular matrix and subcellular stiffness gradients reveal local biomechanical responses.

    Science.gov (United States)

    Tseng, Peter; Di Carlo, Dino

    2014-02-26

    A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

  13. Subcellular Localization of Cadmium in Chlorella vulgaris Beijerinck Strain Bt-09

    Directory of Open Access Journals (Sweden)

    P.B. Lintongan

    2004-06-01

    Full Text Available Growth response curves of Chlorella vulgaris Beijerinck strain Bt-09 to sublethal concentrations of cadmium were evaluated. The growth responses of this microalgal isolate was determined through analysis of chlorophyll a levels. Cadmium was effectively taken up by the cells as determined by Flame Atomic Absorption Spectrophotometry (F-AAS. Subcellular fractionation was undertaken to locate sites that accumulate cadmium.

  14. Subcellular Localization of Galloylated Catechins in Tea Plants (Camellia sinensis (L. O. Kuntze Assessed via Immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Huanhuan eXu

    2016-05-01

    Full Text Available Galloylated catechins, as the main secondary metabolites in the tea plant, including (--epigallocatechin-3-gallate and (--epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilised for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level.

  15. Subcellular location of the helper component-proteinase of Cowpea Aphid-Borne Mosaic Virus

    NARCIS (Netherlands)

    Mlotshwa, S.; Verver, J.; Sithole-Niang, I.; Gopinath, K.; Carette, J.; Kammen, van A.; Wellink, J.

    2002-01-01

    The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of H

  16. Research and teaching with the AFTOL SBD: an informatics resource for fungal subcellular and biochemical data.

    Science.gov (United States)

    Arun Kumar, T K; Blackwell, Meredith; Letcher, Peter M; Roberson, Robert W; McLaughlin, David J

    2013-12-01

    The Structural and Biochemical Database (SBD), developed as part of the US NSF-funded Assembling the Fungal Tree of Life (AFTOL), is a multi-investigator project. It is a major resource to present and manage morphological and biochemical information on Fungi and serves as a phyloinformatics tool for the scientific community. It also is an important resource for teaching mycology. The database, available at http://aftol.umn.edu, includes new and previously published subcellular data on Fungi, supplemented with images and literature links. Datasets automatically combined in NEXUS format from the site permit independent and combined (with molecular data) phylogenetic analyses. Character lists, a major feature of the site, serve as primary reference documents of subcellular and biochemical characters that distinguish taxa across the major fungal lineages. The character lists illustrated with images and drawings are informative for evolutionary and developmental biologists as well as educators, students and the public. Fungal Subcellular Ontology (FSO), developed as part of this effort is a primary initiative to provide a controlled vocabulary describing subcellular structures unique to Fungi. FSO establishes a full complement of terms that provide an operating ontological framework for the database. Examples are provided for using the database for teaching.

  17. A multi-label predictor for identifying the subcellular locations of singleplex and multiplex eukaryotic proteins.

    Directory of Open Access Journals (Sweden)

    Xiao Wang

    Full Text Available Subcellular locations of proteins are important functional attributes. An effective and efficient subcellular localization predictor is necessary for rapidly and reliably annotating subcellular locations of proteins. Most of existing subcellular localization methods are only used to deal with single-location proteins. Actually, proteins may simultaneously exist at, or move between, two or more different subcellular locations. To better reflect characteristics of multiplex proteins, it is highly desired to develop new methods for dealing with them. In this paper, a new predictor, called Euk-ECC-mPLoc, by introducing a powerful multi-label learning approach which exploits correlations between subcellular locations and hybridizing gene ontology with dipeptide composition information, has been developed that can be used to deal with systems containing both singleplex and multiplex eukaryotic proteins. It can be utilized to identify eukaryotic proteins among the following 22 locations: (1 acrosome, (2 cell membrane, (3 cell wall, (4 centrosome, (5 chloroplast, (6 cyanelle, (7 cytoplasm, (8 cytoskeleton, (9 endoplasmic reticulum, (10 endosome, (11 extracellular, (12 Golgi apparatus, (13 hydrogenosome, (14 lysosome, (15 melanosome, (16 microsome, (17 mitochondrion, (18 nucleus, (19 peroxisome, (20 spindle pole body, (21 synapse, and (22 vacuole. Experimental results on a stringent benchmark dataset of eukaryotic proteins by jackknife cross validation test show that the average success rate and overall success rate obtained by Euk-ECC-mPLoc were 69.70% and 81.54%, respectively, indicating that our approach is quite promising. Particularly, the success rates achieved by Euk-ECC-mPLoc for small subsets were remarkably improved, indicating that it holds a high potential for simulating the development of the area. As a user-friendly web-server, Euk-ECC-mPLoc is freely accessible to the public at the website http://levis.tongji.edu.cn:8080/bioinfo

  18. Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS

    Science.gov (United States)

    Nagy, Julia; Grohmann, Dina; Cheung, Alan C. M.; Schulz, Sarah; Smollett, Katherine; Werner, Finn; Michaelis, Jens

    2015-01-01

    The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

  19. Plant nitrogen-use strategy as a driver of rhizosphere archaeal and bacterial ammonia oxidiser abundance.

    Science.gov (United States)

    Thion, Cécile E; Poirel, Jessica D; Cornulier, Thomas; De Vries, Franciska T; Bardgett, Richard D; Prosser, James I

    2016-07-01

    The influence of plants on archaeal (AOA) and bacterial (AOB) ammonia oxidisers (AO) is poorly understood. Higher microbial activity in the rhizosphere, including organic nitrogen (N) mineralisation, may stimulate both groups, while ammonia uptake by plants may favour AOA, considered to prefer lower ammonia concentration. We therefore hypothesised (i) higher AOA and AOB abundances in the rhizosphere than bulk soil and (ii) that AOA are favoured over AOB in the rhizosphere of plants with an exploitative strategy and high N demand, especially (iii) during early growth, when plant N uptake is higher. These hypotheses were tested by growing 20 grassland plants, covering a spectrum of resource-use strategies, and determining AOA and AOB amoA gene abundances, rhizosphere and bulk soil characteristics and plant functional traits. Joint Bayesian mixed models indicated no increase in AO in the rhizosphere, but revealed that AOA were more abundant in the rhizosphere of exploitative plants, mostly grasses, and less abundant under conservative plants. In contrast, AOB abundance in the rhizosphere and bulk soil depended on pH, rather than plant traits. These findings provide a mechanistic basis for plant-ammonia oxidiser interactions and for links between plant functional traits and ammonia oxidiser ecology.

  20. Cooperative adsorption of critical metal ions using archaeal poly-γ-glutamate.

    Science.gov (United States)

    Hakumai, Yuichi; Oike, Shota; Shibata, Yuka; Ashiuchi, Makoto

    2016-06-01

    Antimony, beryllium, chromium, cobalt (Co), gallium (Ga), germanium, indium (In), lithium, niobium, tantalum, the platinoids, the rare-earth elements (including dysprosium, Dy), and tungsten are generally regarded to be critical (rare) metals, and the ions of some of these metals are stabilized in acidic solutions. We examined the adsorption capacities of three water-soluble functional polymers, namely archaeal poly-γ-glutamate (L-PGA), polyacrylate (PAC), and polyvinyl alcohol (PVA), for six valuable metal ions (Co(2+), Ni(2+), Mn(2+), Ga(3+), In(3+), and Dy(3+)). All three polymers showed apparently little or no capacity for divalent cations, whereas L-PGA and PAC showed the potential to adsorb trivalent cations, implying the beneficial valence-dependent selectivity of anionic polyelectrolytes with multiple carboxylates for metal ions. PVA did not adsorb metal ions, indicating that the crucial role played by carboxyl groups in the adsorption of crucial metal ions cannot be replaced by hydroxyl groups under the conditions. In addition, equilibrium studies using the non-ideal competitive adsorption model indicated that the potential for L-PGA to be used for the removal (or collection) of water-soluble critical metal ions (e.g., Ga(3+), In(3+), and Dy(3+)) was far superior to that of any other industrially-versatile PAC materials. PMID:27013333

  1. Archaeal enrichment in the hypoxic zone in the northern Gulf of Mexico.

    Science.gov (United States)

    Gillies, Lauren E; Thrash, J Cameron; deRada, Sergio; Rabalais, Nancy N; Mason, Olivia U

    2015-10-01

    Areas of low oxygen have spread exponentially over the past 40 years, and are cited as a key stressor on coastal ecosystems. The world's second largest coastal hypoxic (≤ 2 mg of O2 l(-1)) zone occurs annually in the northern Gulf of Mexico. The net effect of hypoxia is the diversion of energy flow away from higher trophic levels to microorganisms. This energy shunt is consequential to the overall productivity of hypoxic water masses and the ecosystem as a whole. In this study, water column samples were collected at 39 sites in the nGOM, 21 of which were hypoxic. Analysis of the microbial community along a hypoxic to oxic dissolved oxygen gradient revealed that the relative abundance (iTag) of Thaumarchaeota species 16S rRNA genes (> 40% of the microbial community in some hypoxic samples), the absolute abundance (quantitative polymerase chain reaction; qPCR) of Thaumarchaeota 16S rRNA genes and archaeal ammonia-monooxygenase gene copy number (qPCR) were significantly higher in hypoxic samples. Spatial interpolation of the microbial and chemical data revealed a continuous, shelfwide band of low dissolved oxygen waters that were dominated by Thaumarchaeota (and Euryarchaeota), amoA genes and high concentrations of phosphate in the nGOM, thus implicating physicochemical forcing on microbial abundance. PMID:25818237

  2. Plant nitrogen-use strategy as a driver of rhizosphere archaeal and bacterial ammonia oxidiser abundance.

    Science.gov (United States)

    Thion, Cécile E; Poirel, Jessica D; Cornulier, Thomas; De Vries, Franciska T; Bardgett, Richard D; Prosser, James I

    2016-07-01

    The influence of plants on archaeal (AOA) and bacterial (AOB) ammonia oxidisers (AO) is poorly understood. Higher microbial activity in the rhizosphere, including organic nitrogen (N) mineralisation, may stimulate both groups, while ammonia uptake by plants may favour AOA, considered to prefer lower ammonia concentration. We therefore hypothesised (i) higher AOA and AOB abundances in the rhizosphere than bulk soil and (ii) that AOA are favoured over AOB in the rhizosphere of plants with an exploitative strategy and high N demand, especially (iii) during early growth, when plant N uptake is higher. These hypotheses were tested by growing 20 grassland plants, covering a spectrum of resource-use strategies, and determining AOA and AOB amoA gene abundances, rhizosphere and bulk soil characteristics and plant functional traits. Joint Bayesian mixed models indicated no increase in AO in the rhizosphere, but revealed that AOA were more abundant in the rhizosphere of exploitative plants, mostly grasses, and less abundant under conservative plants. In contrast, AOB abundance in the rhizosphere and bulk soil depended on pH, rather than plant traits. These findings provide a mechanistic basis for plant-ammonia oxidiser interactions and for links between plant functional traits and ammonia oxidiser ecology. PMID:27130939

  3. Geographic Distribution of Archaeal Ammonia Oxidizing Ecotypes in the Atlantic Ocean.

    Science.gov (United States)

    Sintes, Eva; De Corte, Daniele; Haberleitner, Elisabeth; Herndl, Gerhard J

    2016-01-01

    In marine ecosystems, Thaumarchaeota are most likely the major ammonia oxidizers. While ammonia concentrations vary by about two orders of magnitude in the oceanic water column, archaeal ammonia oxidizers (AOA) vary by only one order of magnitude from surface to bathypelagic waters. Thus, the question arises whether the key enzyme responsible for ammonia oxidation, ammonia monooxygenase (amo), exhibits different affinities to ammonia along the oceanic water column and consequently, whether there are different ecotypes of AOA present in the oceanic water column. We determined the abundance and phylogeny of AOA based on their amoA gene. Two ecotypes of AOA exhibited a distribution pattern reflecting the reported availability of ammonia and the physico-chemical conditions throughout the Atlantic, and from epi- to bathypelagic waters. The distinction between these two ecotypes was not only detectable at the nucleotide level. Consistent changes were also detected at the amino acid level. These changes include substitutions of polar to hydrophobic amino acid, and glycine substitutions that could have an effect on the configuration of the amo protein and thus, on its activity. Although we cannot identify the specific effect, the ratio of non-synonymous to synonymous substitutions (dN/dS) between the two ecotypes indicates a strong positive selection between them. Consequently, our results point to a certain degree of environmental selection on these two ecotypes that have led to their niche specialization. PMID:26903961

  4. Potential radiocarbon chronology tool using archaeal tetraether lipids for the western Arctic Ocean sediment

    Science.gov (United States)

    Uchida, M.; Kondo, M.; Utsumi, M.; Shibata, Y.

    2011-12-01

    The Arctic Ocean plays a major role in global climate changes by changing global energy balance through ice-albedo feedback and by affecting the oceanic thermohaline circulation through the water exchange with the Atlantic and Pacific Ocean. For future prediction of those changes in Arctic Ocean it is necessary to reconstruct details of past climate history with accurate age model. However, it is not easy to make age model of sediment cores covering late Pleistocene-Holocene transition including global abrupt climate change because preservation of carbonate fossil such as planktonic foraminifera is very limited. In the glacial time, foraminifera is almost barren. Thus, so far climate history in the Arctic Ocean environment is poorly understood. For these reasons, we are looking for alternative chronological target instead of foraminifera. In this study, radiocarbon contents of archaeal glycerol dibiphytanyl glycerol tetraether lipids (GDGTs) from Arctic surface sediments, which is also used for paleothermometry, were measured and compared with those of dissolved inorganic carbon (DIC) of sea water, planktonic and benthic foraminifera, shell, and bulk organic matter in the same horizons. We will discuss potential as chronology using the GDGTs and carbon sources of these compounds.

  5. Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    The tertiary fold of the elongation factor, aEF-1β, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1β was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1β structure revealed close similarity to its human analogue, eEF-1β. In agreement with studies on EF-Ts and human EF-1β, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1α. aEF-1β was also found to bind calcium in the groove between helix α2 and strand β4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation

  6. Sub-cellular force microscopy in single normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  7. Fast subcellular localization by cascaded fusion of signal-based and homology-based methods

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-10-01

    Full Text Available Abstract Background The functions of proteins are closely related to their subcellular locations. In the post-genomics era, the amount of gene and protein data grows exponentially, which necessitates the prediction of subcellular localization by computational means. Results This paper proposes mitigating the computation burden of alignment-based approaches to subcellular localization prediction by a cascaded fusion of cleavage site prediction and profile alignment. Specifically, the informative segments of protein sequences are identified by a cleavage site predictor using the information in their N-terminal shorting signals. Then, the sequences are truncated at the cleavage site positions, and the shortened sequences are passed to PSI-BLAST for computing their profiles. Subcellular localization are subsequently predicted by a profile-to-profile alignment support-vector-machine (SVM classifier. To further reduce the training and recognition time of the classifier, the SVM classifier is replaced by a new kernel method based on the perturbational discriminant analysis (PDA. Conclusions Experimental results on a new dataset based on Swiss-Prot Release 57.5 show that the method can make use of the best property of signal- and homology-based approaches and can attain an accuracy comparable to that achieved by using full-length sequences. Analysis of profile-alignment score matrices suggest that both profile creation time and profile alignment time can be reduced without significant reduction in subcellular localization accuracy. It was found that PDA enjoys a short training time as compared to the conventional SVM. We advocate that the method will be important for biologists to conduct large-scale protein annotation or for bioinformaticians to perform preliminary investigations on new algorithms that involve pairwise alignments.

  8. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  9. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Science.gov (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  10. Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs.

    Directory of Open Access Journals (Sweden)

    Pedro R Frade

    Full Text Available Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%. About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater, host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.

  11. Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs.

    Science.gov (United States)

    Frade, Pedro R; Roll, Katharina; Bergauer, Kristin; Herndl, Gerhard J

    2016-01-01

    Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity. PMID:26788724

  12. Predicting the subcellular location of apoptosis proteins based on recurrence quantification analysis and the Hilbert-Huang transform

    Institute of Scientific and Technical Information of China (English)

    Han Guo-Sheng; Yu Zu-Guo; Anh Vo

    2011-01-01

    Apoptosis proteins play an important role in the development and homeostasis of an organism.The elucidation of the subcellular locations and functions of these proteins is helpful for understanding the mechanism of programmed cell death.In this paper,the recurrent quantification analysis,Hilbert-Huang transform methods,the maximum relevance and minimum redundancy method and support vector machine are used to predict the subcellular location of apoptosis proteins.The validation of the jackknife test suggests that the proposed method can improve the prediction accuracy of the subcellular location of apoptosis proteins and its application may be promising in other fields.

  13. Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly.

    Science.gov (United States)

    Meshcheryakov, Vladimir A; Wolf, Matthias

    2016-06-01

    Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA-like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP-however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH-FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA-binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC-like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly. PMID:27060465

  14. Bacterial, archaeal and eukaryal community structures throughout soil horizons of harvested and naturally disturbed forest stands.

    Science.gov (United States)

    Hartmann, Martin; Lee, Sangwon; Hallam, Steven J; Mohn, William W

    2009-12-01

    Disturbances caused by timber harvesting have critical long-term effects on the forest soil microbiota and alter fundamental ecosystem services provided by these communities. This study assessed the effects of organic matter removal and soil compaction on microbial community structures in different soil horizons 13 years after timber harvesting at the long-term soil productivity site at Skulow Lake, British Columbia. A harvested stand was compared with an unmanaged forest stand. Ribosomal intergenic spacer profiles of bacteria, archaea and eukarya indicated significantly different community structures in the upper three soil horizons of the two stands, with differences decreasing with depth. Large-scale sequencing of the ribosomal intergenic spacers coupled to small-subunit ribosomal RNA genes allowed taxonomic identification of major microbial phylotypes affected by harvesting or varying among soil horizons. Actinobacteria and Gemmatimonadetes were the predominant phylotypes in the bacterial profiles, with the relative abundance of these groups highest in the unmanaged stand, particularly in the deeper soil horizons. Predominant eukaryal phylotypes were mainly assigned to known mycorrhizal and saprotrophic species of Basidiomycetes and Ascomycetes. Harvesting affected Basidiomycetes to a minor degree but had stronger effects on some Ascomycetes. Archaeal profiles had low diversity with only a few predominant crenarchaeal phylotypes whose abundance appeared to increase with depth. Detection of these effects 13 years after harvesting may indicate a long-term change in processes mediated by the microbial community with important consequences for forest productivity. These effects warrant more comprehensive investigation of the effects of harvesting on the structure of forest soil microbial communities and the functional consequences. PMID:19659501

  15. Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars.

    Science.gov (United States)

    La Duc, Myron T; Vaishampayan, Parag; Nilsson, Henrik R; Torok, Tamas; Venkateswaran, Kasthuri

    2012-08-01

    Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.

  16. Molecular Determinants Responsible for the Subcellular Localization of HSV-1 UL4 Protein

    Institute of Scientific and Technical Information of China (English)

    Wei-wei Pan; Jing Long; Jun-ji Xing; Chun-fu Zheng

    2011-01-01

    The function of the herpes simplex virus type 1(HSV-1)UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study,fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein(EYFP),the nuclear export signals(NES)of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition,the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4.Furthermore,the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1(CRM l)-dependent manner involving RanGTP hydrolysis.

  17. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Subhash Chandra

    2008-05-30

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  18. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin.

    Science.gov (United States)

    Upadhyay, Srijana; Xu, Xinping; Lowry, David; Jackson, Jennifer C; Roberson, Robert W; Lin, Xiaorong

    2016-03-22

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes. PMID:26972005

  19. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

    Directory of Open Access Journals (Sweden)

    Srijana Upadhyay

    2016-03-01

    Full Text Available Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  20. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    Science.gov (United States)

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  1. Fast Fourier Transform-based Support Vector Machine for Subcellular Localization Prediction Using Different Substitution Models

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    There are approximately 109 proteins in a cell. A hotspot in bioinformatics is how to identify a protein's subcellular localization, if its sequence is known. In this paper, a method using fast Fourier transform-based support vector machine is developed to predict the subcellular localization of proteins from their physicochemical properties and structural parameters. The prediction accuracies reached 83% in prokaryotic organisms and 84% in eukaryotic organisms with the substitution model of the c-p-v matrix (c, composition; p, polarity; and v, molecular volume). The overall prediction accuracy was also evaluated using the "leave-one-out" jackknife procedure. The influence of the substitution model on prediction accuracy has also been discussed in the work. The source code of the new program is available on request from the authors.

  2. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: subcellular distribution, chemical forms and thiol pools.

    Science.gov (United States)

    Weng, Bosen; Xie, Xiangyu; Weiss, Dominik J; Liu, Jingchun; Lu, Haoliang; Yan, Chongling

    2012-11-01

    In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  3. Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park.

    Science.gov (United States)

    Bowen De León, Kara; Gerlach, Robin; Peyton, Brent M; Fields, Matthew W

    2013-01-01

    The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park (YNP), Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatures (44°C, 63°C, 75°C). The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi) at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus) at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44 and 63°C springs and a thermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus Nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus Caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different) to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts.

  4. Archaeal and anaerobic methane oxidizer communities in the Sonora Margin cold seeps, Guaymas Basin (Gulf of California).

    Science.gov (United States)

    Vigneron, Adrien; Cruaud, Perrine; Pignet, Patricia; Caprais, Jean-Claude; Cambon-Bonavita, Marie-Anne; Godfroy, Anne; Toffin, Laurent

    2013-08-01

    Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the 'BIG' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel 'ANME-2c Sonora' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support

  5. Controls on bacterial and archaeal community structure and greenhouse gas production in natural, mined, and restored Canadian peatlands

    Directory of Open Access Journals (Sweden)

    Nathan eBasiliko

    2013-07-01

    Full Text Available Northern peatlands are important global C reservoirs, largely because of their slow rates of microbial C mineralization. Particularly in sites that are heavily influenced by anthropogenic disturbances, there is scant information about microbial ecology and whether or not microbial community structure influences greenhouse gas production. This work characterized communities of bacteria and archaea using terminal restriction fragment length polymorphism and sequence analysis of 16S rRNA and functional genes across eight natural, mined, or restored peatlands in two locations in eastern Canada. Correlations were explored among chemical properties of peat, bacterial and archaeal community structure, and carbon dioxide and methane production rates under oxic and anoxic conditions. Bacteria and archaea similar to those found in other peat soil environments were detected. In contrast to other reports, methanogen diversity was low in our study, with only 2 groups of known or suspected methanogens. Although mining and restoration affected substrate availability and microbial activity, these land-uses did not consistently affect bacterial or archaeal community composition. In fact, larger differences were observed between the two locations and between oxic and anoxic peat samples than between mined and restored sites, with anoxic samples characterized by less detectable bacterial diversity and stronger dominance by members of the phylum Acidobacteria. There were also no apparent strong linkages between prokaryote community structure and methane or carbon dioxide production, suggesting that different organisms exhibit functional redundancy and/or that the same taxa function at very different rates when exposed to different peat substrates. In contrast to other earlier work focusing on fungal communities across similar mined and restored peatlands, bacterial and archaeal communities appeared to be more resistant or resilient to peat substrate changes brought

  6. Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park

    Science.gov (United States)

    Bowen De León, Kara; Gerlach, Robin; Peyton, Brent M.; Fields, Matthew W.

    2013-01-01

    The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park (YNP), Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatures (44°C, 63°C, 75°C). The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi) at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus) at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44 and 63°C springs and a thermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus Nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus Caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different) to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts. PMID:24282404

  7. Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park

    Directory of Open Access Journals (Sweden)

    Kara Bowen De León

    2013-11-01

    Full Text Available The Heart Lake Geyser Basin (HLGB is remotely located at the base of Mount Sheridan in southern Yellowstone National Park, Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5 hot springs with varying temperatures (44°C, 63°C, 75°C. The bacterial populations were generally stable over time, but varied by temperature. The dominant bacterial community changed from moderately thermophilic and photosynthetic members (Cyanobacteria and Chloroflexi at 44°C to a mixed photosynthetic and thermophilic community (Deinococcus-Thermus at 63°C and a non-photosynthetic thermophilic community at 75°C. The archaeal community was more variable across time and was predominantly a methanogenic community in the 44°C and 63°C springs and a hyperthermophilic community in the 75°C spring. The 75°C spring demonstrated large shifts in the archaeal populations and was predominantly Candidatus Nitrosocaldus, an ammonia-oxidizing crenarchaeote, in the 2007 sample, and almost exclusively Thermofilum or Candidatus Caldiarchaeum in the 2009 sample, depending on SSU rRNA gene region examined. The majority of sequences were dissimilar (≥10% different to any known organisms suggesting that HLGB possesses numerous new phylogenetic groups that warrant cultivation efforts.

  8. Accumulation, subcellular distribution and toxicity of inorganic mercury and methylmercury in marine phytoplankton

    Energy Technology Data Exchange (ETDEWEB)

    Wu Yun [Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2011-10-15

    We examined the accumulation, subcellular distribution, and toxicity of Hg(II) and MeHg in three marine phytoplankton (the diatom Thalassiosira pseudonana, the green alga Chlorella autotrophica, and the flagellate Isochrysis galbana). For MeHg, the inter-species toxic difference could be best interpreted by the total cellular or intracellular accumulation. For Hg(II), both I. galbana and T. pseudonana exhibited similar sensitivity, but they each accumulated a different level of Hg(II). A higher percentage of Hg(II) was bound to the cellular debris fraction in T. pseudonana than in I. galbana, implying that the cellular debris may play an important role in Hg(II) detoxification. Furthermore, heat-stable proteins were a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). Elucidating the different subcellular fates of Hg(II) and MeHg may help us understand their toxicity in marine phytoplankton at the bottom of aquatic food chains. - Highlights: > The inter-species toxic difference of methylmercury in marine phytoplankton can be explained by its total cellular or intracellular accumulation. > The inter-species toxic difference of inorganic mercury in marine phytoplankton can be explained by its subcellular distribution. > Heat-stable protein was a major binding pool for MeHg, while the cellular debris was an important binding pool for Hg(II). - The inter-species difference in methylmercury and inorganic mercury toxicity in phytoplankton can be explained by cellular accumulation and subcellular distribution.

  9. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

    OpenAIRE

    Furger, AM; Neve, J; Burger, K; Patel, R.; Gullerova, M; Li, W.; Hoque, M.; Tian, B.

    2015-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we employed a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected t...

  10. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells

    OpenAIRE

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-01-01

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Bur...

  11. Quantifying subcellular dynamics in apoptotic cells with two-dimensional Gabor filters

    OpenAIRE

    Pasternack, Robert M.; Rabin, Bryan; Zheng, Jing-Yi; Boustany, Nada N.

    2010-01-01

    We demonstrate an optical Fourier filtering method which can be used to characterize subcellular morphology during dynamic cellular function. In this paper, our Fourier filters were based on two-dimensional Gabor elementary functions, which can be tuned to sense directly object size and orientation. We utilize this method to quantify changes in mitochondrial and nuclear structure during the first three hours of apoptosis. We find that the technique is sensitive to a decrease in particle orien...

  12. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

    Directory of Open Access Journals (Sweden)

    Salih Maysoon

    2009-06-01

    Full Text Available Abstract Background Tail anchored (TA membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP possesses two alternatively spliced tail anchors (TA1 or TA2 but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER, whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  13. Multiple Sequence Elements Facilitate Chp Rho GTPase Subcellular Location, Membrane Association, and Transforming Activity

    OpenAIRE

    Chenette, Emily J.; Mitin, Natalia Y.; Der, Channing J.

    2006-01-01

    Cdc42 homologous protein (Chp) is a member of the Rho family of small GTPases and shares significant sequence and functional similarity with Cdc42. However, unlike classical Rho GTPases, we recently found that Chp depends on palmitoylation, rather than prenylation, for association with cellular membranes. Because palmitoylation alone is typically not sufficient to promote membrane association, we evaluated the possibility that other carboxy-terminal residues facilitate Chp subcellular associa...

  14. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

    OpenAIRE

    Srijana Upadhyay; Xinping Xu; David Lowry; Jennifer C. Jackson; Robert W. Roberson; Xiaorong Lin

    2016-01-01

    Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs, and how it is deposited as extracellular granules, remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are ...

  15. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-01

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  16. *CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

    OpenAIRE

    Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

    2006-01-01

    Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity ...

  17. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels;

    2014-01-01

    , 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca(2+)]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution...... by transmission electron microscopy. At fatigue, tetanic [Ca(2+)]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P

  18. Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene

    OpenAIRE

    Guirimand, Grégory; Guihur, Anthony; Phillips, Michael A.; Oudin, Audrey; Glévarec, Gaëlle; Mahroug, Samira; Melin, Céline; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Rodríguez-Concepción, Manuel; Burlat, Vincent; Courdavault, Vincent

    2012-01-01

    Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple...

  19. Cellular and subcellular localization of protein I in the peripheral nervous system.

    OpenAIRE

    Fried, G.; Nestler, E J; De Camilli, P; Stjärne, L; Olson, L; Lundberg, J M; Hökfelt, T; Ouimet, C C; Greengard, P

    1982-01-01

    The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated tha protein I is present throughout the peripheral nervous system. Denervation studies of adrenal medulla and iris suggested that the protein I contained in peripheral...

  20. Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

    OpenAIRE

    Sengeløv, H.; Kjeldsen, L; Diamond, M S; Springer, T A; Borregaard, N

    1993-01-01

    The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% ...

  1. Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park

    OpenAIRE

    Kara Bowen De León; Robin eGerlach; Peyton, Brent M.; Matthew W Fields

    2013-01-01

    The Heart Lake Geyser Basin (HLGB) is remotely located at the base of Mount Sheridan in southern Yellowstone National Park, Wyoming, USA and is situated along Witch Creek and the northwestern shore of Heart Lake. Likely because of its location, little is known about the microbial community structure of springs in the HLGB. Bacterial and archaeal populations were monitored via small subunit (SSU) rRNA gene pyrosequencing over 3 years in 3 alkaline (pH 8.5) hot springs with varying temperatur...

  2. Archaeal amoA gene diversity points to distinct biogeography of ammonia-oxidizing Crenarchaeota in the ocean

    OpenAIRE

    Sintes, Eva; Bergauer, Kristin; de Corte, Daniele; Yokokawa, Taichi; Herndl, Gerhard J.

    2013-01-01

    Mesophilic ammonia-oxidizing Archaea (AOA) are abundant in a diverse range of marine environments, including the deep ocean, as revealed by the quantification of the archaeal amoA gene encoding the alpha-subunit of the ammonia monooxygenase. Using two different amoA primer sets, two distinct ecotypes of marine Crenarchaeota Group I (MCGI) were detected in the waters of the tropical Atlantic and the coastal Arctic. The HAC-AOA ecotype (high ammonia concentration AOA) was ≍ 8000 times and 15 ti...

  3. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  4. Parasites modify sub-cellular partitioning of metals in the gut of fish

    Energy Technology Data Exchange (ETDEWEB)

    Oyoo-Okoth, Elijah, E-mail: elijaoyoo2009@gmail.com [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Admiraal, Wim [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Osano, Odipo [Division of Environmental Health, School of Environmental Studies, Moi University, P.O. Box 3900, Eldoret (Kenya); Kraak, Michiel H.S. [Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 9424/1090 GE (Netherlands); Gichuki, John; Ogwai, Caleb [Kenya Marine and Fisheries Research Institute, P.O. Box 1881, Kisumu (Kenya)

    2012-01-15

    Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the subcellular fractions of the gut in presence of an endoparasite (Ligula intestinalis). The fish were sampled along four sites in Lake Victoria, Kenya differing in metal contamination. Accumulation of Cd, Cr and Zn was higher in the whole body and in the gut of parasitized fish compared to non-parasitized fish, while Cu was depleted in parasitized fish. Generally, for both non-parasitized and parasitized fish, Cd, Cr and Zn partitioned in the cytosolic fractions and Cu in the particulate fraction. Metal concentrations in organelles within the particulate fractions of the non-parasitized fish were statistically similar except for Cd in the lysosome, while in the parasitized fish, Cd, Cr and Zn were accumulated more by the lysosome and microsomes. In the cytosolic fractions, the non-parasitized fish accumulated Cd, Cr and Zn in the heat stable proteins (HSP), while in the parasitized fish the metals were accumulated in the heat denatured proteins (HDP). On the contrary, Cu accumulated in the HSP in parasitized fish. The present study revealed specific binding of metals to potentially sensitive sub-cellular fractions in fish in the presence of parasites, suggesting interference with metal detoxification, and potentially affecting the health status of fish hosts in Lake Victoria.

  5. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    Science.gov (United States)

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  6. Colocalization of fluorescence and Raman microscopic images for the identification of subcellular compartments: a validation study.

    Science.gov (United States)

    Krauß, Sascha D; Petersen, Dennis; Niedieker, Daniel; Fricke, Inka; Freier, Erik; El-Mashtoly, Samir F; Gerwert, Klaus; Mosig, Axel

    2015-04-01

    A major promise of Raman microscopy is the label-free detailed recognition of cellular and subcellular structures. To this end, identifying colocalization patterns between Raman spectral images and fluorescence microscopic images is a key step to annotate subcellular components in Raman spectroscopic images. While existing approaches to resolve subcellular structures are based on fluorescence labeling, we propose a combination of a colocalization scheme with subsequent training of a supervised classifier that allows label-free resolution of cellular compartments. Our colocalization scheme unveils statistically significant overlapping regions by identifying correlation between the fluorescence color channels and clusters from unsupervised machine learning methods like hierarchical cluster analysis. The colocalization scheme is used as a pre-selection to gather appropriate spectra as training data. These spectra are used in the second part as training data to establish a supervised random forest classifier to automatically identify lipid droplets and nucleus. We validate our approach by examining Raman spectral images overlaid with fluorescence labelings of different cellular compartments, indicating that specific components may indeed be identified label-free in the spectral image. A Matlab implementation of our colocalization software is available at . PMID:25679809

  7. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    Science.gov (United States)

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice. PMID:27450963

  8. Cadmium accumulation and subcellular distribution in relation to cadmium chloride induced cytotoxicity in vitro

    International Nuclear Information System (INIS)

    A bovine kidney cell culture system was used to assess what relationship cadmium (Cd) uptake and subcellular distribution had to cadmium chloride induced cytotoxicity. Twenty-four hour incubation with 0.1-10 μM Cd elicited 0-90% cytotoxicity. Fifty percent cytotoxicity was estimated to result from 0.8 μM Cd. A concentration-related Cd accumulation paralleled the cytotoxicity profile. The time-course for Cd accumulation was linear for the first 6 h of exposure and plateaued by 18 h post-exposure. When the degree of cytotoxicity was compared with the cellular Cd burden at 24 h post-treatment a least-squares linear regression analysis (r=0.93) indicated a direct relationship. Subcellular distribution studies indicated greater than 90% Cd recovery from the soluble supernatant (105,000 g) at all levels of cytotoxicity studied. Metallothionein sequestered less than 25% of the cellular Cd. As a result of the correlation of the degree of cytotoxicity with the cellular Cd burden and the independence of subcellular distribution from cytotoxicity, a cumulative mechanism of toxicity for Cd in MDBK cells was suggested. (author)

  9. KCC2-dependent subcellular ECl difference of ON-OFF retinal ganglion cells in larval zebrafish

    Directory of Open Access Journals (Sweden)

    Rongwei eZhang

    2013-05-01

    Full Text Available Subcellular difference in the reversal potential of Cl- (ECl has been found in many types of neurons. As local ECl largely determines the action of nearby GABAergic/glycinergic synapses, subcellular ECl difference can effectively regulate neuronal computation. The ON-OFF retinal ganglion cell (RGC processes both ON and OFF visual signals via its ON and OFF dendrites, respectively. It is thus interesting to investigate whether the ON and OFF dendrites of single RGCs exhibit different local ECl. Here, using in vivo gramicidin-perforated patch recording in larval zebrafish ON-OFF RGCs, we examine local ECl at the ON and OFF dendrites, and soma through measuring light-evoked ON and OFF inhibitory responses, and GABA-induced response at the soma, respectively. We find there are subcellular ECl differences between the soma and dendrite, as well as between the ON and OFF dendrites of single RGCs. These somato-dendritic and inter-dendritic ECl differences are dependent on the Cl- extruder, K+/Cl- co-transporter (KCC2, because they are largely diminished by down-regulating kcc2 expression with morpholino oligonucleotides or by blocking KCC2 function with furosemide. Thus, our findings indicate that there exists KCC2-dependent ECl difference between the ON and OFF dendrites of individual ON-OFF RGCs that may differentially affect visual processing in the ON and OFF pathways.

  10. Predicting the Subcellular Localization of Human Proteins Using Machine Learning and Exploratory Data Analysis

    Institute of Scientific and Technical Information of China (English)

    George K. Acquaah-Mensah; Sonia M. Leach; Chittibabu Guda

    2006-01-01

    Identifying the subcellular localization of proteins is particularly helpful in the functional annotation of gene products. In this study, we use Machine Learning and Exploratory Data Analysis (EDA) techniques to examine and characterize amino acid sequences of human proteins localized in nine cellular compartments. A dataset of 3,749 protein sequences representing human proteins was extracted from the SWISS-PROT database. Feature vectors were created to capture specific amino acid sequence characteristics. Relative to a Support Vector Machine, a Multi-layer Perceptron, and a Naive Bayes classifier, the C4.5 Decision Tree algorithm was the most consistent performer across all nine compartments in reliably predicting the subcellular localization of proteins based on their amino acid sequences (average Precision=0.88; average Sensitivity=0.86). Furthermore, EDA graphics characterized essential features of proteins in each compartment. As examples,proteins localized to the plasma membrane had higher proportions of hydrophobic amino acids; cytoplasmic proteins had higher proportions of neutral amino acids;and mitochondrial proteins had higher proportions of neutral amino acids and lower proportions of polar amino acids. These data showed that the C4.5 classifier and EDA tools can be effective for characterizing and predicting the subcellular localization of human proteins based on their amino acid sequences.

  11. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

    Directory of Open Access Journals (Sweden)

    Xia eGuo

    2016-01-01

    Full Text Available Histone deacetylases (HDACs are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2 and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12 remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  12. Birth and rapid subcellular adaptation of a hominoid-specific CDC14 protein.

    Directory of Open Access Journals (Sweden)

    Lia Rosso

    2008-06-01

    Full Text Available Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18-25 million years ago (Mya. CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7-12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function.

  13. A workflow for mathematical modeling of subcellular metabolic pathways in leaf metabolism of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eNägele

    2013-12-01

    Full Text Available During the last decade genome sequencing has experienced a rapid technological development resulting in numerous sequencing projects and applications in life science. In plant molecular biology, the availability of sequence data on whole genomes has enabled the reconstruction of metabolic networks. Enzymatic reactions are predicted by the sequence information. Pathways arise due to the participation of chemical compounds as substrates and products in these reactions. Although several of these comprehensive networks have been reconstructed for the genetic model plant Arabidopsis thaliana, the integration of experimental data is still challenging. Particularly the analysis of subcellular organization of plant cells limits the understanding of regulatory instances in these metabolic networks in vivo. In this study, we develop an approach for the functional integration of experimental high-throughput data into such large-scale networks. We present a subcellular metabolic network model comprising 524 metabolic intermediates and 548 metabolic interactions derived from a total of 2769 reactions. We demonstrate how to link the metabolite covariance matrix of different Arabidopsis thaliana accessions with the subcellular metabolic network model for the inverse calculation of the biochemical Jacobian, finally resulting in the calculation of a matrix which satisfies a Lyaponov equation involving a covariance matrix. In this way, differential strategies of metabolite compartmentation and involved reactions were identified in the accessions when exposed to low temperature.

  14. Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

    DEFF Research Database (Denmark)

    Greber, Basil J; Boehringer, Daniel; Godinic-Mikulcic, Vlatka;

    2012-01-01

    additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter...... thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared...... between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes...

  15. Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs.

    Science.gov (United States)

    Fischer, Martin A; Güllert, Simon; Neulinger, Sven C; Streit, Wolfgang R; Schmitz, Ruth A

    2016-01-01

    The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis

  16. Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes.

    Science.gov (United States)

    Makarova, Kira S; Wolf, Yuri I; Forterre, Patrick; Prangishvili, David; Krupovic, Mart; Koonin, Eugene V

    2014-09-01

    Microbial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote "dark matter islands", in archaeal genomes. The dark matter islands comprise up to 20% of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures.

  17. Temperature and pH dependence of DNA ejection from archaeal lemon-shaped virus His1.

    Science.gov (United States)

    Hanhijärvi, K J; Ziedaite, G; Hæggström, E; Bamford, D H

    2016-07-01

    The archaeal virus His1 isolated from a hypersaline environment infects an extremely halophilic archaeon Haloarcula hispanica. His1 features a lemon-shaped capsid, which is so far found only in archaeal viruses. This unique capsid can withstand high salt concentrations, and can transform into a helical tube, which in turn is resistant to extremely harsh conditions. Hypersaline environments exhibit a wide range of temperatures and pH conditions, which present an extra challenge to their inhabitants. We investigated the influence of pH and temperature on DNA ejection from His1 virus using single-molecule fluorescence experiments. The observed number of ejecting viruses is constant in pH 5 to 9, while the ejection process is suppressed at pH below 5. Similarly, the number of ejections within 15-42 °C shows only a minor increase around 25-37 °C. The maximum velocity of single ejected DNA increases with temperature, in qualitative agreement with the continuum model of dsDNA ejection. PMID:26820561

  18. Archaeal and bacterial diversity in two hot springs from geothermal regions in Bulgaria as demostrated by 16S rRNA and GH-57 genes.

    Science.gov (United States)

    Stefanova, Katerina; Tomova, Iva; Tomova, Anna; Radchenkova, Nadja; Atanassov, Ivan; Kambourova, Margarita

    2015-12-01

    Archaeal and bacterial diversity in two Bulgarian hot springs, geographically separated with different tectonic origin and different temperature of water was investigated exploring two genes, 16S rRNA and GH-57. Archaeal diversity was significantly higher in the hotter spring Levunovo (LV) (82°C); on the contrary, bacterial diversity was higher in the spring Vetren Dol (VD) (68°C). The analyzed clones from LV library were referred to twenty eight different sequence types belonging to five archaeal groups from Crenarchaeota and Euryarchaeota. A domination of two groups was observed, Candidate Thaumarchaeota and Methanosarcinales. The majority of the clones from VD were referred to HWCG (Hot Water Crenarchaeotic Group). The formation of a group of thermophiles in the order Methanosarcinales was suggested. Phylogenetic analysis revealed high numbers of novel sequences, more than one third of archaeal and half of the bacterial phylotypes displayed similarity lower than 97% with known ones. The retrieved GH-57 gene sequences showed a complex phylogenic distribution. The main part of the retrieved homologous GH-57 sequences affiliated with bacterial phyla Bacteroidetes, Deltaproteobacteria, Candidate Saccharibacteria and affiliation of almost half of the analyzed sequences is not fully resolved. GH-57 gene analysis allows an increased resolution of the biodiversity assessment and in depth analysis of specific taxonomic groups. [Int Microbiol 18(4):217-223 (2015)].

  19. Distribution of bacterial and archaeal ether lipids in soils and surface sediments of Tibetan lakes: Implications for GDGT-based proxies in saline high mountain lakes

    NARCIS (Netherlands)

    Günther, F.; Thiele, A.; Gleixner, G.; Xu, B.Q.; Yao, T.; Schouten, S.

    2014-01-01

    Bacterial and archaeal lipids, such as glycerol dialkyl glycerol tetraethers (GDGTs) and dialkyl glycerol diethers, are increasingly used as proxies for specific environmental parameters, such as air temperature and soil pH in lacustrine environments. Little is known, however, about the distribution

  20. Effect of supplementing coconut or krabok oil, rich in medium-chain fatty acids on ruminal fermentation, protozoa and archaeal population of bulls

    NARCIS (Netherlands)

    Panyakaew, P.; Boon, N.; Goel, G.; Yuangklang, C.; Schonewille, J.T.; Hendriks, W.H.; Fievez, V.

    2013-01-01

    Medium-chain fatty acids (MCFA), for example, capric acid (C10:0), myristic (C14:0) and lauric (C12:0) acid, have been suggested to decrease rumen archaeal abundance and protozoal numbers. This study aimed to compare the effect of MCFA, either supplied through krabok (KO) or coconut (CO) oil, on rum

  1. The subcellular compartmentalization of arginine metabolizing enzymes and their role in endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Feng eChen

    2013-07-01

    Full Text Available The endothelial production of nitric oxide (NO mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle proliferation and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS through the conversion of its substrate, L-arginine to L-citrulline. Reduced access to L-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2 metabolize L-arginine to generate L-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of L-arginine. Indeed, supplemental L-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis suggesting additional mechanisms. The compartmentalization of intracellular L-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of L-arginine. Indeed the subcellular location of L-arginine metabolizing enzymes plays important functional roles. In endothelial cells, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localtion. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, ASL, co-localize with eNOS and facilitate NO release. This review highlights the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease.

  2. Subcellular Accumulation of Cadmium in Corn and Wheat Plants at Different Levels of Phosphorus

    Institute of Scientific and Technical Information of China (English)

    YANG; ZHENGSHAOJIAN; 等

    1999-01-01

    Corn and wheat plants were grown in a nutrient culture solution at four levels of phosphorus (0,0.12,0.60 and 3.0mmol L-1) and two levels of cadmium(0 and 4.0umol L-1) in greenhouse for a 18-day period.The concentrations of phosphorus and cadmium in cell wall,cytoplasm and vacuoles of roots and leaves were examined by cell fractionation techniques.With increasing phosphorus in medium,the contents of P in cell wall,cytoplasm and vacuoles of corn and wheat roots and leaves increased.The highest content of P was observed in cell wall,next in vacuoles,and the lowest in cytoplasm.The wheat subcellular fractions in both roots and leaves hab higher concentrations of phosphorus than those of corn.Increasing phosphorus in medium significantly inhibited the intracellular Cd accumulation in both species,However,at P concentration up to 3.0mmol L-1,the Cd content in cell wall was increased.Increasing phosphorus resulted in reduction of the subcellular Cd content in cell wall was increased.Increasing phosphorus resulted in reduction of the subcellualr Cd content in corn and wheat leaves.Compared with corn,the wheat roots had a higher Cd content in the cell wall and vacuoles and a lower in cytoplasm,while in leaf subcellular fractions the wheat cell had a higher Cd content in its vacuoles and a lower one in its cytoplasm,The results indicate that phosphorus may be involved in sequestration of Cd ionic activity in both cell wall and vaculoes by forming insoluble Cd phosphate.

  3. Linking sub-cellular biomarkers to embryo aberrations in the benthic amphipod Monoporeia affinis.

    Science.gov (United States)

    Reutgard, Martin; Furuhagen, Sara

    2016-04-01

    To adequately assess and monitor environmental status in the aquatic environment a broad approach is needed that integrates physical variables, chemical analyses and biological effects at different levels of the biological organization. Embryo aberrations in the Baltic Sea key species Monoporeia affinis can be induced by both metals and organic substances as well as by hypoxia, increasing temperatures and malnutrition. This amphipod has therefore been used for more than three decades as a biological effect indicator in monitoring and assessment of chemical pollution and environmental stress. However, little is known about the sub-cellular mechanisms underlying embryo aberrations. An improved mechanistic understanding may open up the possibility of including sub-cellular alterations as sensitive warning signals of stress-induced embryo aberrations. In the present study, M. affinis was exposed in microcosms to 4 different sediments from the Baltic Sea. After 88-95 days of exposure, survival and fecundity were determined as well as the frequency and type of embryo aberrations. Moreover, oxygen radical absorption capacity (ORAC) was assayed as a proxy for antioxidant defense, thiobarbituric acid reactive substances (TBARS) level as a measure of lipid peroxidation and acetylcholinesterase (AChE) activity as an indicator of neurotoxicity. The results show that AChE and ORAC can be linked to the frequency of malformed embryos and arrested embryo development. The occurrence of dead broods was significantly associated with elevated TBARS levels. It can be concluded that these sub-cellular biomarkers are indicative of effects that could affect Darwinian fitness and that oxidative stress is a likely mechanism in the development of aberrant embryos in M. affinis.

  4. Multiple Features Based Two-stage Hybrid Classifier Ensembles for Subcellular Phenotype Images Classification

    Directory of Open Access Journals (Sweden)

    Bailing Zhang - China

    2010-12-01

    Full Text Available Subcellular localization is a key functional characteristic of proteins. As an interesting ``bio-image informatics'' application, an automatic, reliable and efficient prediction system for protein subcellular localization can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, we propose a two-stage multiple classifier system to improve classification reliability by introducing rejection option. The system is built as a cascade of two classifier ensembles. The first ensemble consists of set of binary SVMs which generalizes to learn a general classification rule and the second ensemble, which also include three distinct classifiers, focus on the exceptions rejected by the rule. A new way to induce diversity for the classifier ensembles is proposed by designing classifiers that are based on descriptions of different feature patterns. In addition to the Subcellular Location Features (SLF generally adopted in earlier researches, three well-known texture feature descriptions have been applied to cell phenotype images, which are the local binary patterns (LBP, Gabor filtering and Gray Level Coocurrence Matrix (GLCM. The different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. Using the public benchmark 2D HeLa cell images, a high classification accuracy 96% is obtained with rejection rate $21\\%$ from the proposed system by taking advantages of the complementary strengths of feature construction and majority-voting based classifiers' decision fusions.

  5. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC imaging method for cadmium ions (Cd2+ was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  6. Brain glycogen – new perspectives on its metabolic function and regulation at the subcellular level

    Directory of Open Access Journals (Sweden)

    Linea Frimodt Obel

    2012-03-01

    Full Text Available Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g. liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies – it is a highly dynamic molecule with versatile implications in brain function, i.e. synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on i the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP, ii alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and iii a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g. turnover is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  7. Clinical significance of subcellular localization of KL-6 mucin in primary colorectal adenocarcinoma and metastatic tissues

    Institute of Scientific and Technical Information of China (English)

    Qian Guo; Masatoshi Makuuchi; Wei Tang; Yoshinori Inagaki; Yutaka Midorikawa; Norihiro Kokudo; Yasuhiko Sugawara; Munehiro Nakata; Toshiro Konishi; Hirokazu Nagawa

    2006-01-01

    AIM: To assess subcellular localization of KL-6 mucin and its clinicopathological significance in colorectal carcinoma as well as metastatic lymph node and liver tissues.METHODS: Colorectal carcinoma tissues as well as metastatic lymph node and liver tissues were collected from 82 patients who underwent colorectomy or hepatectomy. Tissues were subjected to immunohistochemical analysis using KL-6 antibody.RESULTS: Of the 82 colorectal carcinoma patients, 6showed no staining, 29 showed positive staining only in the apical membrane, and 47 showed positive staining in the circumferential membrane and/or cytoplasm.Positive staining was not observed in non-cancerous colorectal epithelial cells surrounding the tumor tissues.The five-year survival rate was significantly lower in cases showing positive staining in the circumferential membrane and/or cytoplasm (63.0%) than those showing positive staining only in the apical membrane (85.7%) and those showing no staining (100%).Statistical analysis between clinicopathological factors and subcellular localization of KL-6 mucin showed that KL-6 localization in the circumferential membrane and/or cytoplasm was significantly associated with the presence of venous invasion (P=0.0003), lymphatic invasion (P<0.0001), lymph node metastasis (P<0.0001),liver metastasis (P=0.058), and advanced histological stage (P<0.0001). Positive staining was observed in all metastatic lesions tested as well as in the primary colorectal carcinoma tissues.CONCLUSION: The subcellular staining pattern of KL-6 in colorectal adenocarcinoma may be an important indicator for unfavorable behaviors such as lymph node and liver metastasis, as well as for the prognosis of patients.

  8. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    Science.gov (United States)

    Zhang, Yu; Mi, Lan; Xiong, Rongling; Wang, Pei-Nan; Chen, Ji-Yao; Yang, Wuli; Wang, Changchun; Peng, Qian

    2009-07-01

    Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs) in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  9. Self-organization of the MinE ring in subcellular Min oscillations

    OpenAIRE

    Derr, Julien; Hopper, Jason T.; Sain, Anirban; Rutenberg, Andrew D.

    2009-01-01

    We model the self-organization of the MinE ring that is observed during subcellular oscillations of the proteins MinD and MinE within the rod-shaped bacterium {\\it Escherichia coli}. With a steady-state approximation, we can study the MinE-ring generically -- apart from the other details of the Min oscillation. Rebinding of MinE to depolymerizing MinD filament tips controls MinE ring formation through a scaled cell shape parameter $\\tilde{r}$. We find two types of E-ring profiles near the fil...

  10. A signal separation technique for sub-cellular imaging using dynamic optical coherence tomography

    CERN Document Server

    Ammari, Habib; Shi, Cong

    2016-01-01

    This paper aims at imaging the dynamics of metabolic activity of cells. Using dynamic optical coherence tomography, we introduce a new multi-particle dynamical model to simulate the movements of the collagen and the cell metabolic activity and develop an efficient signal separation technique for sub-cellular imaging. We perform a singular-value decomposition of the dynamic optical images to isolate the intensity of the metabolic activity. We prove that the largest eigenvalue of the associated Casorati matrix corresponds to the collagen. We present several numerical simulations to illustrate and validate our approach.

  11. Subcellular localization of SUN2 is regulated by lamin a and Rab5

    OpenAIRE

    Liang, Ying; Chiu, Peng Hang; Yip, Kit Yan; Chan, Siu Yuen

    2011-01-01

    SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the n...

  12. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    Directory of Open Access Journals (Sweden)

    Chen Ji-Yao

    2009-01-01

    Full Text Available Abstract Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  13. Mapping the Microscale Origins of MRI Contrast with Subcellular NV Diamond Magnetometry

    CERN Document Server

    Davis, Hunter C; Bhatnagar, Aadyot; Lee-Gosselin, Audrey; Barry, John F; Glenn, David R; Walsworth, Ronald L; Shapiro, Mikhail G

    2016-01-01

    Magnetic resonance imaging (MRI) is a widely used biomedical imaging modality that derives much of its contrast from microscale magnetic field gradients in biological tissues. However, the connection between these sub-voxel field patterns and MRI contrast has not been studied experimentally. Here, we describe a new method to map subcellular magnetic fields in mammalian cells and tissues using nitrogen vacancy diamond magnetometry and connect these maps to voxel-scale MRI contrast, providing insights for in vivo imaging and contrast agent design.

  14. Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

    OpenAIRE

    Harhaj, Edward W; Sun, Shao-Cong

    1999-01-01

    Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between ...

  15. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    OpenAIRE

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintain...

  16. The influence of different land uses on the structure of archaeal communities in Amazonian anthrosols based on 16S rRNA and amoA genes.

    Science.gov (United States)

    Taketani, Rodrigo Gouvêa; Tsai, Siu Mui

    2010-05-01

    Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use. PMID:20204349

  17. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    Science.gov (United States)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  18. Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.

    Science.gov (United States)

    Cafasso, Jacquelyn; Manjasetty, Babu A; Karr, Elizabeth A; Sandman, Kathleen; Chance, Mark R; Reeve, John N

    2010-11-01

    TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Å resolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V(M)) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein. PMID:21045304

  19. Preliminary Crystallography Confirms that the Archaeal DNA-binding and Tryptophan-sensing Regulator TrpY is a Dimer

    Energy Technology Data Exchange (ETDEWEB)

    J Cafasso; B Manjasetty; E Karr; K Sandman; M Chance; J Reeve

    2011-12-31

    TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 87, c = 147 {angstrom}, and diffracted to 2.9 {angstrom} resolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V{sub M}) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein.

  20. A dimeric Rep protein initiates replication of a linear archaeal virus genome: implications for the Rep mechanism and viral replication

    DEFF Research Database (Denmark)

    Oke, Muse; Kerou, Melina; Liu, Huanting;

    2011-01-01

    The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a...... active-site tyrosine and the 5' end of the DNA, releasing a 3' DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely...... positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral...

  1. A shift in the archaeal nitrifier community in response to natural and anthropogenic disturbances in the northern Gulf of Mexico.

    Science.gov (United States)

    Newell, Silvia E; Eveillard, Damien; McCarthy, Mark J; Gardner, Wayne S; Liu, Zhanfei; Ward, Bess B

    2014-02-01

    The Gulf of Mexico is affected by hurricanes and suffers seasonal hypoxia. The Deepwater Horizon oil spill impacted every trophic level in the coastal region. Despite their importance in bioremediation and biogeochemical cycles, it is difficult to predict the responses of microbial communities to physical and anthropogenic disturbances. Here, we quantify sediment ammonia-oxidizing archaeal (AOA) community diversity, resistance and resilience, and important geochemical factors after major hurricanes and the oil spill. Dominant AOA archetypes correlated with different geochemical factors, suggesting that different AOA are constrained by distinct parameters. Diversity was lowest after the hurricanes, showing weak resistance to physical disturbances. However, diversity was highest during the oil spill and coincided with a community shift, suggesting a new alternative stable state sustained for at least 1 year. The new AOA community was not significantly different from that at the spill site 1 year after the spill. This sustained shift in nitrifier community structure may be a result of oil exposure. PMID:24596268

  2. Phylogenetic and Functional Analysis of Metagenome Sequence from High-Temperature Archaeal Habitats Demonstrate Linkages between Metabolic Potential and Geochemistry

    DEFF Research Database (Denmark)

    Inskeep, William P; Jay, Zackary J; Herrgard, Markus;

    2013-01-01

    Geothermal habitats in Yellowstone National Park (YNP) provide an unparalleled opportunity to understand the environmental factors that control the distribution of archaea in thermal habitats. Here we describe, analyze, and synthesize metagenomic and geochemical data collected from seven high......-temperature sites that contain microbial communities dominated by archaea relative to bacteria. The specific objectives of the study were to use metagenome sequencing to determine the structure and functional capacity of thermophilic archaeal-dominated microbial communities across a pH range from 2.5 to 6.......4 and to discuss specific examples where the metabolic potential correlated with measured environmental parameters and geochemical processes occurring in situ. Random shotgun metagenome sequence (∼40-45 Mb Sanger sequencing per site) was obtained from environmental DNA extracted from high-temperature sediments and...

  3. Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Morimoto, Kazuhiro

    2013-01-01

    Azithromycin (AZM), a 15-membered ring macrolide antimicrobial agent, has an antibacterial spectrum that includes intracellular parasitic pathogens that survive or intracellularly multiply in alveolar macrophages (AMs). The subcellular distribution of AZM in AMs was evaluated in vitro in comparison with clarithromycin (CAM). AZM and CAM (50 µM) were applied to the NR8383 cells, used as an in vitro model of AMs, followed by incubation at 37°C or 4°C. The total amount of AZM in cells and subcellular distribution (cell fractionation) was determined after incubation. High level of AZM accumulation was observed in the NR8383 cells at 37°C, and the equilibrium intracellular to extracellular concentration ratio (I/E ratio) was approximately 680, which was remarkably higher than that of CAM (equilibrium I/E ratio=28). The intracellular accumulation of AZM and CAM was temperature dependent. In addition, AZM distributed to the granules fraction including organelles and soluble fraction including cytosol in the NR8383 cells, whereas CAM mainly distributed in soluble fraction. The amount of AZM in the granules fraction was markedly reduced in the presence of ammonium chloride for increase in intracellular pH. These results indicate that AZM is distributed in acidic compartment in AMs. This study suggests that high AZM accumulation in the NR8383 cells is due to the trapping and/or binding in acidic organelles, such as lysosomes.

  4. SUBCELLULAR LOCALIZATION OF BETA CATENIN IN COLORECTAL NON NEOPLASTIC AND NEOPLASTIC LESIONS

    Directory of Open Access Journals (Sweden)

    Diah Rini Handjari

    2011-12-01

    Full Text Available Loss of adenomatous polyposis coli (APC function is typically an early event in sporadic colorectal cancer (CRC pathogenesis. The key tumor suppressor function of the APC protein lies in its ability to destabilize free cytoplasmic beta catenin. This lead to the accumulation of nuclear beta catenin, and together with the DNA binding protein Tcf-4, function as a transcriptional activator. Accumulation of stabilized free β-catenin is considered as an early event and perhaps initiating the process in intestinal tumorigenesis. Neoplastic transformation in the CRC associated chronic colitis is considered similar to the adenoma-carcinoma sequence in sporadic CRC. The distinguish feature from the CRC-related colitis is the difference in time and frequency changes. Loss of APC function, regarded as the beginning of a very common event in sporadic CRC, but the CRC associated chronic colitis generally occurs at the end of thedysplasia-carcinoma sequence. This research was conducted to determine the subcellular location of beta catenin expression in chronic colitis, colorectal adenomas and carcinomas that were evaluated by immunohistochemical staining. It can be concluded that beta-catenin is a component that plays a role in the development of the CRC and the subcellular location of beta-catenin can describe its oncogenic activity.

  5. Zn Accumulation and Subcellular Distribution in the Zn Hyperaccumulator Sedum alfredii Hance

    Institute of Scientific and Technical Information of China (English)

    LI Ting-Qiang; YANG Xiao-E; YANG Jin-Yan; HE Zhen-Li

    2006-01-01

    Zn accumulation and subcellular distribution in leaves of the hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum alfredii Hance were studied using radiotracer and gradient centrifugation techniques. Leaf Zn accumulation in the HE of S. alfredii was 18.5-26.7 times greater than that in the NHE when the plants were grown at 1-500μmol Zn L-1. Leaf section uptake of 65Zn was highly dependent on external Zn levels. Greater 65Zn uptake in HE was noted only at external Zn levels ≥ 100μmol L-1. Zinc subcellular distribution in the leaves of the two ecotypes of S. alfredii was: cell wall > soluble fraction > cell organelle. However, more Zn was distributed to the leaf cell wall and soluble fractions for HE than for NHE. In the leaf of HE, 91%-94% of the Zn was found in the cell walls and the soluble fraction and only 6%-9% Zn was distributed in the cell organelle fraction. For NHE, about 20%-26% Zn was recovered in the cell organelle fraction. In stems, Zn distribution to the cell wall fraction was approximately two fold greater in the HE than that in the NHE. For the hyperaccumulating ecotype of S. alfredii, the cell wall and the vacuole played a very important role in Zn tolerance and hyperaccumulation.

  6. Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in Hep-2 cells

    Institute of Scientific and Technical Information of China (English)

    Min CHEN; Ping JIANG

    2004-01-01

    AIM: To study the effects of actinomycin D on subcellular distribution of nucleolar protein fibrillarin in HEp-2(human esophageal epithelial type 2) cells, and molecular mechanisms for maintenance of fibrillarin in nucleolus.METHODS: Indirect immunofiuorescence assay was employed to investigate subcellular distribution of nucleolar protein fibrillarin and immunoblotting analysis was used to detect the total cellular amount of fibrillarin. RESULTS:Control cells with no drug treatment showed bright clumpy nucleolar staining, which indicated that fibrillarin decorated the nucleolus only. Treatment with actinomycin D caused dislocation of fibrillarin from nucleoli to nucleoplasm with numerous stained small nucleoplasmic entities. Immunoblotting showed that neither total cellular amount of fibrillarin nor the integrity of fibrillarin was changed upon the treatment. The dislocation of fibrillarin in cells treated at a lower concentration (0.05 mg/L) of actinomycin D, was totally reversible after removal of the drug from the medium. However, this reversion was not observed at a high drug concentration (1 mg/L). CONCLUSION:Actinomycin D induced dislocation of fibrillarin from nucleoli to nucleoplasm in HEp-2 cells. The retention of fibrillarin within the nucleolus was related to active RNA synthesis.

  7. Subcellular localization of several heavy metals of Hg,Cd and Pb in human liver

    Institute of Scientific and Technical Information of China (English)

    CHEN Chunying; ZHANG Peiqun; CHAI Zhifang

    2005-01-01

    Liver, as an important metabolic and detoxicological organ of human body, can be used as a good bioindicator for evaluating body burden of environmental pollutants. Its elemental contents and their chemical forms are closely related to the status of human health and disease. In this paper, the liver samples collected from normal subjects were separated to different subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol by differential centrifugation. Then their concentrations of heavy metals of As, Pb, Cd, and Hg were determined by atomic absorption and atomic fluorescent spectroscopy. Our results show no significant difference with literature ones when comparing their gross concentrations. In the case of their subcellular distribution, the Hg concentrations are higher in mitochondrial, microsomal and cytosolic fractions; the Cd concentrations are higher in cytosolic and mitochondrial fractions, while As highest in nuclear fraction. The highest concentration of Pb is found in microsomal fraction with similarity to Fe. Mercury in liver is mainly in the form of inorganic, and methylmercury ranged from 9% to 50% with the average value of 20.9%(13.3%. These results indicate that the cellular distribution and the accumulated target organelles are quite different among these heavy metals, which suggest their various pathways and toxic mechanism in vivo.

  8. Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

    Institute of Scientific and Technical Information of China (English)

    Petra Talapka; Nikolett Bódi; Izabella Battonyai; (E)va Fekete; Mária Bagyánszki

    2011-01-01

    AIM:To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS:Postembedding immunoelectronmicroscopy was performed,in which primary antibodies for neuronal NOS (nNOS),endothelial NOS (eNOS),and inducible NOS (iNOS),were visualized with protein A-gold-conjugated secondary antibodies.Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera.The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS:Postembedding immunoelectronmicroscopy revealed the presence of nNOS,eNOS,and iNOS immunoreactivity in the myenteric neurons,the enteric smooth muscle cells,and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum.The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed.In the control experiments,in which the primary antiserum was omitted,virtually no postembedding gold particles were observed. CONCLUSION:This postembedding immunoelectronmicroscopic study provided the first evidence of celltype- specific differences in the subcellular distributions of NOS isoforms.

  9. Radioimmunoassay of steroids in homogenates and subcellular fractions of testicular tissue

    International Nuclear Information System (INIS)

    Radioimmunoassays for testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha, 17beta-diol (DIOL) in homogenates of whole testis, interstitial tissue and seminiferous tubules as well as subcellular fractions of the latter were developed. Steroids were extracted with acetone, submitted to several solvent partitions and isolated by a celite: propylene glycol: ethylene glycol column chromatography. Anit-T serum was used for the assay of T and DTH, and a specific anti-Diol serum for DIOL. Subcellular fractions were separated by differential centrifugation. The nuclear fraction was purified by centrifugation in a dense sucrose buffer followed by several washings. Losses were corrected according to recovery of DNA. Optimal conditions for purification of acetone extracts at minimal losses were established. Validation of the method was studied testing linear regression of logit-log transformations of standard curves and parallelism with unknowns. T was the steroid present in higher concentrations in all samples studied. It is concluded that the present method for determination of endogenous androgen concentrations in testicular tissue is valid and might be useful in studing testicular function. (orig.)

  10. Sequential changes of serum and liver subcellular oxidants and antoxidant concentrations in silymarin treated male rats

    Directory of Open Access Journals (Sweden)

    J.A.A. Al-Sa'aidi

    2016-04-01

    Full Text Available The present study aimed to investigate the role of silymarin as an antioxidant and/or it activity in induction of the endogenous antioxidants in intact adult male rats. Seventy males were randomly devided into control and silymarin treated groups (35 each, and were drenched with drinking water and silymarin suspension (200 mg/ kg b.w daily for 40 days. Each group was allocated to 5 equal subgroups; sacrificed before treatment (0 day, and after 10, 20, 30, and 40 days of treatment. At the end of each period, males were anaesthesized, dissected and blood samples were obtained for assessment of MDA, SOD, CAT and GSH concentrations. liver samples (1 g have been removed and homogenized for assessment of liver subcellular MDA, SOD, CAT and GSH concentrations. At the end of each periods, serum and liver subcellular MAD concentrations showed no significant changes between groups, whereas SOD, CAT, and GSH concentrations significantly increased at 10, 20, 30, and 40 day periods in silymarin treated males compared with control. It can be concluded that silymarin antioxidant activity is of pharmacological value not only as an antioxidant by itself but also as an inducer of endogenous enzymatic and non-enzymatic antioxidants even in normal intact male.

  11. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    Science.gov (United States)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  12. Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

    Science.gov (United States)

    Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

    2015-11-01

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

  13. Subcellular localization and rearrangement of endoplasmic reticulum by Brome mosaic virus capsid protein.

    Science.gov (United States)

    Bamunusinghe, Devinka; Seo, Jang-Kyun; Rao, A L N

    2011-03-01

    Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, as well as in other positive-strand RNA viruses pathogenic to humans (e.g., poliovirus) and animals (e.g., Flock House virus), is functionally coupled to replication. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. In this study, the application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a green fluorescent protein (GFP) fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV-infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP itself has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. The significance of CP-induced vesicles in relation to CP-organized viral functions that are linked to replication-coupled packaging is discussed.

  14. Slit2 expression and its correlation with subcellular localization of β-catenin in gastric cancer.

    Science.gov (United States)

    Shi, Rongliang; Liu, Weiyan; Liu, Bingya; Xu, Ziping; Chen, Liping; Zhang, Ziping

    2013-10-01

    Gastric cancer is the fourth most common cancer worldwide. Several signaling pathways are involved in gastric cancer development and progression. Slit2 was recently found to be involved in cancer; however, its expression pattern in gastric cancer has not been discovered yet. In the present study, we investigated the expression of Slit2 in human gastric cancer and its correlation with the expression and subcellular localization of β-catenin. Immunohistochemistry (IHC) staining revealed that Slit2 was highly expressed in human gastric cancer tissues, while it was low or weakly expressed in normal gastric tissues. The differences in clinicopathological features between different groups were determined using Pearson's χ2 test. Slit2 levels were significantly associated with differentiation, Lauren's classification, lymph node metastasis and TNM staging. Slit2 levels were positively correlated with β-catenin level in gastric cancer tissues and cell lines. High levels of Slit2 were correlated with the membrane localization of β-catenin, and low levels of Slit2 were correlated with nuclear translocation of β-catenin in both gastric cancer tissues and cell lines assayed by IHC and immunofluorescence staining, respectively. Our data suggest that Slit2 was highly expressed in gastric cancer patients with less advanced clinicopathological features. Slit2 levels were correlated with β-catenin level and subcellular localization.

  15. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: exposure to cadmium.

    Science.gov (United States)

    Wang, Meng-Jiao; Wang, Wen-Xiong

    2011-01-25

    The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd(2+)], 0.01-84nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd(2+)] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd(2+)] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC(2) being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs' detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd(2+)] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.

  16. Role of Glucokinase in the Subcellular Localization of Glucokinase Regulatory Protein

    Directory of Open Access Journals (Sweden)

    Ling Jin

    2015-04-01

    Full Text Available Glucokinase (GCK is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein, GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

  17. Subcellular Distribution of Gallium-67 and other Auger-emitting Radionuclides in Human and Animal Tissues

    International Nuclear Information System (INIS)

    The subcellular distributions of the Auger-emitting radiopharmaceuticals 67Ga citrate, 111In-bleomycin, 63Zn-bleomycin, 123/125/I-Conray and 123/125I-Biligram in human and animal tumours and normal tissues were studied using differential centrifugation. For 67Ga, at 19 to 54 h after injection, the observed subcellular distribution patterns were similar in all the tissues studied with 2-5% of the total tissue radioactivity being associated with the cell nuclei. For 63Zn and 111In administered as the bleomycin complex or as chloride, as well as for the X ray contrast media 135/125I-Conray and 123/125I-Biligram the fraction associated with the nuclei was only 1-2%. The studies indicate that, for the 67Ga, 111In, 63Zn complexes or compounds studied, only γ1 to 4% of the Auger emissions are likely to arise in the cell nucleus. (author)

  18. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    Science.gov (United States)

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

  19. Mutational analyses of the signals involved in the subcellular location of DSCR1

    Directory of Open Access Journals (Sweden)

    Henrique-Silva Flávio

    2002-09-01

    Full Text Available Abstract Background Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR, to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human. Results The protein's location was preferentially nuclear, independently of the isoform, cell line and insertion in the GFP's N- or C-terminal. A segment in the C-terminal, which is important in the location of the protein, was identified by deletion. On the other hand, site-directed mutational analyses have indicated the involvement of some serine and threonine residues in this event. Conclusion In this paper, we discuss the identification of amino acids which can be important for subcellular location of DSCR1. The involvement of residues that are prone to phosphorylation suggests that the location and function of DSCR1 may be regulated by kinases and/or phosphatases.

  20. Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis

    Directory of Open Access Journals (Sweden)

    Cheevadhanarak Supapon

    2009-09-01

    Full Text Available Abstract The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

  1. β-amylase in developing apple fruits: activities, amounts and subcellular localization

    Institute of Scientific and Technical Information of China (English)

    张大鹏; 王永章

    2002-01-01

    Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β-amylase is considered one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. The present experiment showed that β-amylase activity was progressively increasing concomitantly with decreasing starch concentrations during apple (Malus domestica Borkh cv. Starkrimson) fruit development. The apparent amount of β-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The subcellular-localization studies via immunogold electron-microscopy technique showed that β-amylase visualized by gold particles was predominantly located in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments. These data proved for the first time that the enzyme is compartmented in its functional sites in plant living cells. The predominantly plastid-distributed pattern of β-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (β-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that β-amylase is involved in starch hydrolysis in plastids of the fruit cells.

  2. Transport and subcellular distribution characteristics of human erythrocyte glucose transporter fused in adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Inho.

    1990-01-01

    Purified human erythrocyte glucose transporters (HEGT) were incorporated into the rat epididymal adipocytes by polyethylene glycol (PEG)-induced fusion. The incorporation of HEGT was found to be dependent on both the molecular weight and the concentration of PEG. Optimal incorporation of HEGT occurred when 10% PEG 8000 was used. This incorporation was found to be proportional to the increasing amounts of HEGT used. Morphology tests showed very little surface adsorption of HEGT on adipocytes after fusion. Transport activity of fused HEGT was studied by measuring equilibrium exchange of 3-O-methylglucose. In adipocytes employed this fusion protocols, the transport rate increased significantly compared with cells treated under non-fusion conditions. This fold increase was directly proportional to the amount of HEGT recovered in the plasma membrane (PM). Furthermore, calculated turnover number of HEGT in fused adipocytes was as high as that of the native adipocyte glucose transporter (AGT). Subcellular distribution of HEGT in fused adipocytes was assessed using ({sup 3}H)cytochalasin B-labeled HEGT vesicles. HEGT was distributed in each subcellular organelle at specific ratios. This relative distribution was almost constant regardless of the amount of HEGT used. The distributions of lipid-labeled HEGT, fluid-phase endocytosed HEGT and free HEGT mixed to adipocyte homogenate were shown to be completely different from that of protein-labeled HEGT fusion.

  3. Transport and subcellular distribution characteristics of human erythrocyte glucose transporter fused in adipocytes

    International Nuclear Information System (INIS)

    Purified human erythrocyte glucose transporters (HEGT) were incorporated into the rat epididymal adipocytes by polyethylene glycol (PEG)-induced fusion. The incorporation of HEGT was found to be dependent on both the molecular weight and the concentration of PEG. Optimal incorporation of HEGT occurred when 10% PEG 8000 was used. This incorporation was found to be proportional to the increasing amounts of HEGT used. Morphology tests showed very little surface adsorption of HEGT on adipocytes after fusion. Transport activity of fused HEGT was studied by measuring equilibrium exchange of 3-O-methylglucose. In adipocytes employed this fusion protocols, the transport rate increased significantly compared with cells treated under non-fusion conditions. This fold increase was directly proportional to the amount of HEGT recovered in the plasma membrane (PM). Furthermore, calculated turnover number of HEGT in fused adipocytes was as high as that of the native adipocyte glucose transporter (AGT). Subcellular distribution of HEGT in fused adipocytes was assessed using [3H]cytochalasin B-labeled HEGT vesicles. HEGT was distributed in each subcellular organelle at specific ratios. This relative distribution was almost constant regardless of the amount of HEGT used. The distributions of lipid-labeled HEGT, fluid-phase endocytosed HEGT and free HEGT mixed to adipocyte homogenate were shown to be completely different from that of protein-labeled HEGT fusion

  4. Soil bacterial and archaeal communities of the Stringer Creek Watershed in relation to soil moisture, chemistry, and gas fluxes

    Science.gov (United States)

    Jones, R. T.; Du, Z.; Riveros-Iregui, D.; Dore, J. E.; Emanuel, R. E.; McGlynn, B. L.; McDermott, T.; Li, X.

    2013-12-01

    The Stringer Creek watershed within the Tenderfoot Creek Experimental Forest (Montana) is a highly instrumented watershed with long-term hydrologic and gas flux measurements, and is an ideal study system to incorporate microbiological characterizations into landscape scale hydrological and biogeochemical studies. As a first attempt to determine how hydrological processes, soil chemistry, and gas fluxes are correlated with bacterial and archaeal lineages in soil, we collected soil samples across the watershed (July 9 - 11, 2012) and used barcoded high-throughput DNA sequencing to characterize the bacterial and archaeal communities. Soils were collected adjacent to gas well sites at 5 cm, 20 cm, and 50 cm depths, corresponding to the depths of the wells. Gas measurements included CO2, CH4, O2, and N2O; soil measurements included water content, % carbon, and % nitrogen. We analyzed 775,000 16S rRNA gene sequences from 28 soil samples. Relative abundances of certain microbial lineages or groups (e.g. methanotrophs, methanogens, Acidobacteria, Bacteroidetes, Firmicutes, Proteobacteria, etc.) varied significantly with CO2, CH4, and O2 concentrations. Furthermore, beta-diversity analyses showed that microbial community composition was significantly governed by water content, % nitrogen, and % carbon; community composition also significantly varied with CO2, CH4, and O2 concentrations. Together, our results suggest that soil environmental factors such as water content, % carbon, and % nitrogen affect microbial community composition, and that microbial community composition correlates with CO2, O2, and CH4 concentrations. Future work will focus on characterizing microbial communities across the entire summer season as soil conditions drastically change from fully saturated to very dry.

  5. Specific bacterial, archaeal, and eukaryotic communities in tidal-flat sediments along a vertical profile of several meters.

    Science.gov (United States)

    Wilms, Reinhard; Sass, Henrik; Köpke, Beate; Köster, Jürgen; Cypionka, Heribert; Engelen, Bert

    2006-04-01

    The subsurface of a tidal-flat sediment was analyzed down to 360 cm in depth by molecular and geochemical methods. A community structure analysis of all three domains of life was performed using domain-specific PCR followed by denaturing gradient gel electrophoresis analysis and sequencing of characteristic bands. The sediment column comprised horizons easily distinguishable by lithology that were deposited in intertidal and salt marsh environments. The pore water profile was characterized by a subsurface sulfate peak at a depth of about 250 cm. Methane and sulfate profiles were opposed, showing increased methane concentrations in the sulfate-free layers. The availability of organic carbon appeared to have the most pronounced effect on the bacterial community composition in deeper sediment layers. In general, the bacterial community was dominated by fermenters and syntrophic bacteria. The depth distribution of methanogenic archaea correlated with the sulfate profile and could be explained by electron donor competition with sulfate-reducing bacteria. Sequences affiliated with the typically hydrogenotrophic Methanomicrobiales were present in sulfate-free layers. Archaea belonging to the Methanosarcinales that utilize noncompetitive substrates were found along the entire anoxic-sediment column. Primers targeting the eukaryotic 18S rRNA gene revealed the presence of a subset of archaeal sequences in the deeper part of the sediment cores. The phylogenetic distance to other archaeal sequences indicates that these organisms represent a new phylogenetic group, proposed as "tidal-flat cluster 1." Eukarya were still detectable at 360 cm, even though their diversity decreased with depth. Most of the eukaryotic sequences were distantly related to those of grazers and deposit feeders.

  6. Analysis of Bacterial and Archaeal Communities along a High-Molecular-Weight Polyacrylamide Transportation Pipeline System in an Oil Field

    Directory of Open Access Journals (Sweden)

    Cai-Yun Li

    2015-04-01

    Full Text Available Viscosity loss of high-molecular-weight partially hydrolyzed polyacrylamide (HPAM solution was observed in a water injection pipeline before being injected into subterranean oil wells. In order to investigate the possible involvement of microorganisms in HPAM viscosity loss, both bacterial and archaeal community compositions of four samples collected from different points of the transportation pipeline were analyzed using PCR-amplification of the 16S rRNA gene and clone library construction method together with the analysis of physicochemical properties of HPAM solution and environmental factors. Further, the relationship between environmental factors and HPAM properties with microorganisms were delineated by canonical correspondence analysis (CCA. Diverse bacterial and archaeal groups were detected in the four samples. The microbial community of initial solution S1 gathered from the make-up tank is similar to solution S2 gathered from the first filter, and that of solution S3 obtained between the first and the second filter is similar to that of solution S4 obtained between the second filter and the injection well. Members of the genus Acinetobacter sp. were detected with high abundance in S3 and S4 in which HPAM viscosity was considerably reduced, suggesting that they likely played a considerable role in HPAM viscosity loss. This study presents information on microbial community diversity in the HPAM transportation pipeline and the possible involvement of microorganisms in HPAM viscosity loss and biodegradation. The results will help to understand the microbial community contribution made to viscosity change and are beneficial for providing information for microbial control in oil fields.

  7. Spatial distribution of archaeal and bacterial ammonia oxidizers in the littoral buffer zone of a nitrogen-rich lake

    Institute of Scientific and Technical Information of China (English)

    Yu Wang; Guibing Zhu; Lei Ye; Xiaojuan Feng; Huub J. M. Op den Camp; Chengqing Yin

    2012-01-01

    The spatial distribution and diversity of archaeal and bacterial ammonia oxidizers (AOA and AOB) were evaluated targeting amoA genes in the gradient of a littoral buffer zone which has been identified as a hot spot for N cycling.Here we found high spatial heterogeneity in the nitrification rate and abundance of ammonia oxidizers in the five sampling sites.The bacterial amoA gene was numerically dominant in most of the surface soil but decreased dramatically in deep layers.Higher nitrification potentials were detected in two sites near the land/water interface at 4.4-6.1 μg NO2--N/(g dry weight soil.hr),while only 1.0-1.7 μg NO2- -N/(gdry weight soil·hr) was measured at other sites.The potential nitrification rates were proportional to the amoA gene abundance for AOB,hut with no significant correlation with AOA.The NH4+ concentration was the most determinative parameter for the abundance of AOB and potential nitrification rates in this study.Higher richness in the surface layer was found in the analysis of biodiversity.Phylogenetic analysis revealed that most of the bacterial amoA sequences in surface soil were affiliated with the genus of Nitrosopira while the archaeal sequences were almost equally affiliated with Candidatus ‘Nitrososphaera gargensis' and Candidatus ‘Nitrosoealdus yellowstonii'.The spatial distribution of AOA and AOB indicated that bacteria may play a more important role in nitrification in the littoral buffer zone of a N-rich lake.

  8. Archaeal diversity and the extent of iron and manganese pyritization in sediments from a tropical mangrove creek (Cardoso Island, Brazil)

    Science.gov (United States)

    Otero, X. L.; Lucheta, A. R.; Ferreira, T. O.; Huerta-Díaz, M. A.; Lambais, M. R.

    2014-06-01

    Even though several studies on the geochemical processes occurring in mangrove soils and sediments have been performed, information on the diversity of Archaea and their functional roles in these ecosystems, especially in subsurface environments, is scarce. In this study, we have analyzed the depth distribution of Archaea and their possible relationships with the geochemical transformations of Fe and Mn in a sediment core from a tropical mangrove creek, using 16S rRNA gene profiling and sequential extraction of different forms of Fe and Mn. A significant shift in the archaeal community structure was observed in the lower layers (90-100 cm), coinciding with a clear decrease in total organic carbon (TOC) content and an increase in the percentage of sand. The comparison of the archaeal communities showed a dominance of methanogenic Euryarchaeota in the upper layers (0-20 cm), whereas Crenarchaeota was the most abundant taxon in the lower layers. The dominance of methanogenic Euryarchaeota in the upper layer of the sediment suggests the occurrence of methanogenesis in anoxic microenvironments. The concentrations of Fe-oxyhydroxides in the profile were very low, and showed positive correlation with the concentrations of pyrite and degrees of Fe and Mn pyritization. Additionally, a partial decoupling of pyrite formation from organic matter concentration was observed, suggesting excessive Fe pyritization. This overpyritization of Fe can be explained either by the anoxic oxidation of methane by sulfate and/or by detrital pyrite tidal transportation from the surrounding mangrove soils. The higher pyritization levels observed in deeper layers of the creek sediment were also in agreement with its Pleistocenic origin.

  9. An ensemble classifier for eukaryotic protein subcellular location prediction using gene ontology categories and amino acid hydrophobicity.

    Directory of Open Access Journals (Sweden)

    Liqi Li

    Full Text Available With the rapid increase of protein sequences in the post-genomic age, it is challenging to develop accurate and automated methods for reliably and quickly predicting their subcellular localizations. Till now, many efforts have been tried, but most of which used only a single algorithm. In this paper, we proposed an ensemble classifier of KNN (k-nearest neighbor and SVM (support vector machine algorithms to predict the subcellular localization of eukaryotic proteins based on a voting system. The overall prediction accuracies by the one-versus-one strategy are 78.17%, 89.94% and 75.55% for three benchmark datasets of eukaryotic proteins. The improved prediction accuracies reveal that GO annotations and hydrophobicity of amino acids help to predict subcellular locations of eukaryotic proteins.

  10. How a (subcellular coincidence detection mechanism featuring layer-5 pyramidal cells may help produce various visual phenomena

    Directory of Open Access Journals (Sweden)

    Talis eBachmann

    2015-12-01

    Full Text Available Perceptual phenomena such as spatio-temporal illusions and masking are typically explained by psychological (cognitive processing theories or large-scale neural theories involving inter-areal connectivity and neural circuits comprising of hundreds or more interconnected single cells. Subcellular mechanisms are hardly used for such purpose. Here a mechanistic theoretical view is presented on how a subcellular brain mechanism of integration of presynaptic signals that arrive at different compartments of layer-5 pyramidal neurons could explain a couple of spatiotemporal visual-phenomenal effects unfolding along very brief time intervals within the range of sub-second temporal scale.

  11. Community Composition and Abundance of Bacterial, Archaeal and Nitrifying Populations in Savanna Soils on Contrasting Bedrock Material in Kruger National Park, South Africa

    Science.gov (United States)

    Rughöft, Saskia; Herrmann, Martina; Lazar, Cassandre S.; Cesarz, Simone; Levick, Shaun R.; Trumbore, Susan E.; Küsel, Kirsten

    2016-01-01

    Savannas cover at least 13% of the global terrestrial surface and are often nutrient limited, especially by nitrogen. To gain a better understanding of their microbial diversity and the microbial nitrogen cycling in savanna soils, soil samples were collected along a granitic and a basaltic catena in Kruger National Park (South Africa) to characterize their bacterial and archaeal composition and the genetic potential for nitrification. Although the basaltic soils were on average 5 times more nutrient rich than the granitic soils, all investigated savanna soil samples showed typically low nutrient availabilities, i.e., up to 38 times lower soil N or C contents than temperate grasslands. Illumina MiSeq amplicon sequencing revealed a unique soil bacterial community dominated by Actinobacteria (20–66%), Chloroflexi (9–29%), and Firmicutes (7–42%) and an increase in the relative abundance of Actinobacteria with increasing soil nutrient content. The archaeal community reached up to 14% of the total soil microbial community and was dominated by the thaumarchaeal Soil Crenarchaeotic Group (43–99.8%), with a high fraction of sequences related to the ammonia-oxidizing genus Nitrosopshaera sp. Quantitative PCR targeting amoA genes encoding the alpha subunit of ammonia monooxygenase also revealed a high genetic potential for ammonia oxidation dominated by archaea (~5 × 107 archaeal amoA gene copies g−1 soil vs. mostly < 7 × 104 bacterial amoA gene copies g−1 soil). Abundances of archaeal 16S rRNA and amoA genes were positively correlated with soil nitrate, N and C contents. Nitrospira sp. was detected as the most abundant group of nitrite oxidizing bacteria. The specific geochemical conditions and particle transport dynamics at the granitic catena were found to affect soil microbial communities through clay and nutrient relocation along the hill slope, causing a shift to different, less diverse bacterial and archaeal communities at the footslope. Overall, our

  12. Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

    Science.gov (United States)

    Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

    1995-08-01

    Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles

  13. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    Science.gov (United States)

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  14. Subcellular localization of PS1 based on PS1/GFP fusing protein

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer's disease (EOFAD). The fusion genes, GFP-PS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT Ⅱ (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution.

  15. Subcellular localization of KL-6 mucin in intraductal papillary mucinous neoplasm of the pancreas.

    Science.gov (United States)

    Inagaki, Yoshinori; Seyama, Yasuji; Hasegawa, Kiyoshi; Tang, Wei; Kokudo, Norihiro

    2014-08-01

    This study aimed to clarify the expression profile of KL-6 mucin in intraductal papillary mucinous neoplasm (IPMN) and its relation to tumor malignancy. Expression of KL-6 mucin in 38 IPMNs (intraductal papillary mucinous adenoma (IPMA), 24 cases; minimally invasive intraductal papillary mucinous carcinoma (MI-IPMC), 8 cases; invasive carcinoma originating from IPMC (IC-IPMC), 6 cases) and 66 pancreatic ductal adenocarcinomas (PDACs) was evaluated immunohistochemically. IC-IPMCs and MI-IPMCs had positive staining of KL-6 mucin whereas 58% of IPMAs tested negative. Subcellular localization of KL-6 mucin varied among IPMNs whereas all of the PDAC had positive expression in the circumferential membrane and cytoplasm of cancer cells. IC-IPMCs and MI-IPMCs had a higher frequency of circumferential membrane and cytoplasmic localization of KL-6 mucin than did IPMAs. These results suggest that localization of KL-6 mucin could be used to predict the malignancy of IPMN. PMID:25047009

  16. Spatial and temporal changes in Bax subcellular localization during NPe6-PDT-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Xing, Da; Chen, Wei R.; Wan, Qingling; Zhou, Feifan

    2008-02-01

    Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Bax, a member of the Bcl-2 family of proteins, is an essential regulator of apoptosis. Bax is normally found in the cytosol of healthy cells, and translocates to mitochondria in response to many apoptotic stimuli. In this study, using real-time single-cell analysis, we have investigated the kinetics of Bax distribution during NPe6-induced apoptosis in ASTC-a-1 cells. In order to monitor Bax subcellular distribution, cells were stained with GFP-Bax and Mito Tracker Red. The results show that Bax redistribution occurred at about 170 min after treated with NPe6-PDT, and then sequestered into clusters associated with the mitochondira within 30 min. Our data clearly showed the spatial and temporal changes in Bax distribution in living cells during NPe6-induced apoptosis.

  17. Expression and subcellular localization of mechano-growth factor in osteoblasts under mechanical stretch

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Mechano-growth factor (MGF) is a stretch sensitive factor in myocytes, and it might also be produced by other mechanocytes under mechanical stimulation. In this study, both the mRNA and protein expression of MGF were detected in stretched osteoblasts. Quantitative analysis showed that a cyclic stretching stimulation caused a quick and sharp increase of MGF mRNA and protein expression from a low basal level under no stretch; the mRNA and protein levels respectively peaked in 6 and 12 h to 5 and 5.2 fold over the basal level and returned to normal by 24 h. The subcellular distribution of MGF protein was revealed by immunofluorescence analysis to be restricted to the nucleus. We concluded that cyclic stretching stimulation could induce MGF expression in osteoblasts in a pulsing fashion; and the nuclear distribution of MGF suggested that MGF might act in mechanocytes as an autocrine growth factor.

  18. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.

    Science.gov (United States)

    Giesen, Charlotte; Wang, Hao A O; Schapiro, Denis; Zivanovic, Nevena; Jacobs, Andrea; Hattendorf, Bodo; Schüffler, Peter J; Grolimund, Daniel; Buhmann, Joachim M; Brandt, Simone; Varga, Zsuzsanna; Wild, Peter J; Günther, Detlef; Bodenmiller, Bernd

    2014-04-01

    Mass cytometry enables high-dimensional, single-cell analysis of cell type and state. In mass cytometry, rare earth metals are used as reporters on antibodies. Analysis of metal abundances using the mass cytometer allows determination of marker expression in individual cells. Mass cytometry has previously been applied only to cell suspensions. To gain spatial information, we have coupled immunohistochemical and immunocytochemical methods with high-resolution laser ablation to CyTOF mass cytometry. This approach enables the simultaneous imaging of 32 proteins and protein modifications at subcellular resolution; with the availability of additional isotopes, measurement of over 100 markers will be possible. We applied imaging mass cytometry to human breast cancer samples, allowing delineation of cell subpopulations and cell-cell interactions and highlighting tumor heterogeneity. Imaging mass cytometry complements existing imaging approaches. It will enable basic studies of tissue heterogeneity and function and support the transition of medicine toward individualized molecularly targeted diagnosis and therapies. PMID:24584193

  19. Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry

    DEFF Research Database (Denmark)

    Björn, Erik; Nygren, Yvonne; Nguyen, Tam T. T. N.;

    2007-01-01

    A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines...... were used. To keep the sample consumption to approximately 10 microl and obtain a high robustness of the system, a flow injection sample introduction system with a 4.6-microl sample loop was used in combination with a conventional pneumatic nebulizer and a spray chamber. The system was optimized...... with respect to signal/noise ratio using a multivariate experimental design. The system proved to be well suited for routine analysis of large sample series, and several hundreds of samples could be analyzed without maintenance or downtime. The detection limit of the method was 0.12 pg (26 pg/g) platinum...

  20. MECHANISMS OF DAMAGING EFFECT OF MANGENESE IN TOXIC CONCENTRATIONS ON CELLULAR AND SUBCELLULAR LEVELS

    Directory of Open Access Journals (Sweden)

    Goncharenko A. V.

    2012-11-01

    Full Text Available Influence of subtoxic concentration of manganese chloride in dose equal to LD 50 on condition of plasmatic membranes (model: erythrocytes and functional activity of cell power (model: the isolated liver mitochondrion of rats was studied. It was established that manganese chloride in fixed concentration caused authentic augmentation of sorption capacity of erythrocytes towards alcian blue, influenced increasing of their spontaneous haemolysis and activation of peroxide oxidation of lipids. In experiment on the isolated mitochondrion it was proved that manganese chloride caused dissociation of an oxidizing phosphorusling and complete inhibition of respiration in concentrations of 3 and 4,5mM. These dependences testify that subtoxic concentration of manganese can damage the cell energy. Thus, this pilot research indicated damaging effect of manganese on cellular (erythrocytes and subcellular (mitochondrion levels which are realized through external functioning of membrane structures and deprived them from restoration.

  1. Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus1

    Science.gov (United States)

    De Luca, Vincenzo; Cutler, Adrian J.

    1987-01-01

    The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline. PMID:16665811

  2. Measurement of subcellular texture by optical Gabor-like filtering with a digital micromirror device

    Science.gov (United States)

    Pasternack, Robert M.; Qian, Zhen; Zheng, Jing-Yi; Metaxas, Dimitris N.; White, Eileen; Boustany, Nada N.

    2010-01-01

    We demonstrate an optical Fourier processing method to quantify object texture arising from subcellular feature orientation within unstained living cells. Using a digital micromirror device as a Fourier spatial filter, we measured cellular responses to two-dimensional optical Gabor-like filters optimized to sense orientation of nonspherical particles, such as mitochondria, with a width around 0.45 μm. Our method showed significantly rounder structures within apoptosis-defective cells lacking the proapoptotic mitochondrial effectors Bax and Bak, when compared with Bax/Bak expressing cells functional for apoptosis, consistent with reported differences in mitochondrial shape in these cells. By decoupling spatial frequency resolution from image resolution, this method enables rapid analysis of nonspherical submicrometer scatterers in an under-sampled large field of view and yields spatially localized morphometric parameters that improve the quantitative assessment of biological function. PMID:18830354

  3. Sub-cellular structure studied by combined atomic force-fluorescence microscopy

    Science.gov (United States)

    Trache, Andreea

    2009-03-01

    A novel experimental technique that integrates atomic force microscopy (AFM) with fluorescence imaging was used to study the role of extracellular matrix proteins in cellular organization. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, we developed a new technology able to investigate cellular behavior at sub-cellular level that integrates an AFM with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. Live smooth muscle cells exhibited differences in focal adhesions and actin pattern depending on the extracellular matrix used for substrate coating. Data obtained by using the AFM-optical imaging integrated technique offer novel quantitative information that allows understanding the fundamental processes of cellular reorganization in response to extracellular matrix modulation. The integrated microscope presented here is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells.

  4. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.;

    2008-01-01

    YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy....... YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial...... origin may be related to cell motility and cell-cell adhesion, features associated with invasion and migration potential of tumor cells Udgivelsesdato: 2008...

  5. The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex.

    Science.gov (United States)

    Bachelard, H S

    1967-07-01

    1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.

  6. Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation.

    Science.gov (United States)

    Jackson, C; Dench, J E; Hall, D O; Moore, A L

    1979-07-01

    Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.The ratio of protein content of the washed mitochondria compared to that in the purified preparation was 7 for spinach and respiratory activity was retained. The physiological integrity and oxidative properties of washed and gradient mitochondria are compared. PMID:16660904

  7. Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses.

    Science.gov (United States)

    Wienkoop, Stefanie; Weiss, Julia; May, Patrick; Kempa, Stefan; Irgang, Susann; Recuenco-Munoz, Luis; Pietzke, Matthias; Schwemmer, Thorsten; Rupprecht, Jens; Egelhofer, Volker; Weckwerth, Wolfram

    2010-06-01

    In the era of fast genome sequencing a critical goal is to develop genome-wide quantitative molecular approaches. Here, we present a metaproteogenomic strategy to integrate proteomics and metabolomics data for systems level analysis in the recently sequenced unicellular green algae Chlamydomonas reinhardtii. To achieve a representative proteome coverage we analysed different growth conditions with protein prefractionation and shotgun proteomics. For protein identification, different genome annotations as well as new gene model predictions with stringent peptide filter criteria were used. An overlapping proteome coverage of 25%, consistent for all databases, was determined. The data are stored in a public mass spectral reference database ProMEX (http://www.promexdb.org/home.shtml). A set of proteotypic peptides comprising Calvin cycle, photosynthetic apparatus, starch synthesis, glycolysis, TCA cycle, carbon concentrating mechanisms (CCM) and other pathways was selected from this database for targeted proteomics (Mass Western). Rapid subcellular fractionation in combination with targeted proteomics allowed for measuring subcellular protein concentrations in attomole per 1000 cells. From the same samples metabolite concentrations and metabolic fluxes by stable isotope incorporation were analyzed. Differences were found in the growth-dependent crosstalk of chloroplastidic and mitochondrial metabolism. A Mass Western survey of all detectable carbonic anhydrases partially involved in carbon-concentrating mechanism (CCM) revealed highest internal cell concentrations for a specific low-CO2-inducible mitochondrial CAH isoform. This indicates its role as one of the strongest CO2-responsive proteins in the crosstalk of air-adapted mixotrophic chloroplast and mitochondrial metabolism in Chlamydomonas reinhardtii. PMID:20358043

  8. Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants12

    Science.gov (United States)

    Corpas, Francisco J.; Barroso, Juan B.; Carreras, Alfonso; Quirós, Miguel; León, Ana M.; Romero-Puertas, María C.; Esteban, Francisco J.; Valderrama, Raquel; Palma, José M.; Sandalio, Luisa M.; Gómez, Manuel; del Río, Luis A.

    2004-01-01

    The cellular and subcellular localization of endogenous nitric oxide (NO˙) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO˙ was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO˙, with g = 2.05 and aN = 12.8 G, was detected in peroxisomes. By fluorometry, NO˙ was also found in these organelles, and the level measured of NO˙ was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO˙ from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO˙ mg−1 protein min−1; was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO˙ could be involved in the process of senescence of pea leaves. PMID:15347796

  9. Abnormal subcellular distribution of GLUT4 protein in obese and insulin-treated diabetic female dogs

    Directory of Open Access Journals (Sweden)

    Vargas A.M.

    2004-01-01

    Full Text Available The GLUT4 transporter plays a key role in insulin-induced glucose uptake, which is impaired in insulin resistance. The objective of the present study was to investigate the tissue content and the subcellular distribution of GLUT4 protein in 4- to 12-year-old control, obese and insulin-treated diabetic mongrel female dogs (4 animals per group. The parametrial white adipose tissue was sampled and processed to obtain both plasma membrane and microsome subcellular fractions for GLUT4 analysis by Western blotting. There was no significant difference in glycemia and insulinemia between control and obese animals. Diabetic dogs showed hyperglycemia (369.9 ± 89.9 mg/dl. Compared to control, the plasma membrane GLUT4, reported per g tissue, was reduced by 55% (P < 0.01 in obese dogs, and increased by 30% (P < 0.05 in diabetic dogs, and the microsomal GLUT4 was increased by ~45% (P < 0.001 in both obese and diabetic animals. Considering the sum of GLUT4 measured in plasma membrane and microsome as total cellular GLUT4, percent GLUT4 present in plasma membrane was reduced by ~65% (P < 0.001 in obese compared to control and diabetic animals. Since insulin stimulates GLUT4 translocation to the plasma membrane, percent GLUT4 in plasma membrane was divided by the insulinemia at the time of tissue removal and was found to be reduced by 75% (P < 0.01 in obese compared to control dogs. We conclude that the insulin-stimulated translocation of GLUT4 to the cell surface is reduced in obese female dogs. This probably contributes to insulin resistance, which plays an important role in glucose homeostasis in dogs.

  10. Identification and subcellular localization of molecular complexes of Gq/11α protein in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Zdenka Drastichova; Jiri Novotny

    2012-01-01

    Heterotrimeric G-proteins localized in the plasma membrane convey the signals from G-protein-coupled receptors (GPCRs) to different effectors.At least some types of Gprotein o subunits have been shown to be partly released from plasma membranes and to move into the cytosol after receptor activation by the agonists.However,the mechanism underlying subcellular redistribution of trimeric G-proteins is not well understood and no definitive conclusions have been reached regarding the translocation of Gα subunits between membranes and cytosol.Here we used subcellular fractionation and clear-native polyacrylamide gel electrophoresis to identify molecular complexes of Gq/1 1α protein and to determine their localization in isolated fractions and stability in naive and thyrotropin-releasing hormone (TRH)-treated HEK293 cells expressing high levels of TRH receptor and G11α protein.We identified two high-molecular-weight complexes of 300 and 140 kDa in size comprising the Gq/11 protein,which were found to be membrane-bound.Both of these complexes dissociated after prolonged treatment with TRH.Still other Gq/11α protein complexes of lower molecular weight were determined in the cytosol.These 70 kDa protein complexes were barely detectable under control conditions but their levels markedly increased after prolonged (4-16 h)hormone treatment.These results support the notion that a portion of Gq/11α can undergo translocation from the membrane fraction into soluble fraction after a long-term activation of TRH receptor.At the same time,these findings indicate that the redistribution of Gq/11α is broughtabout by the dissociation of high-molecular-weight complexes and concomitant formation of low-molecular-weight complexes containing the Gq/11α protein.

  11. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash

    2004-06-15

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes {sup 13}C and {sup 15}N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK{sub 1} kidney cells at mass 28 ({sup 13}C{sup 15}N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of {sup 39}K, {sup 23}Na and {sup 40}Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  12. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    Science.gov (United States)

    Chandra, Subhash

    2004-06-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  13. In situ spatiotemporal mapping of flow fields around seeded stem cells at the subcellular length scale.

    Directory of Open Access Journals (Sweden)

    Min Jae Song

    Full Text Available A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms.

  14. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Arieh Riskin

    2015-10-01

    Full Text Available Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC in culture. Methods: Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC. To study GLUT1 targeting and recycling in living mouse MEC (MMEC in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM, or exposed to secretion medium (SM, containing prolactin. Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes. Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

  15. SMYD3 interacts with HTLV-1 Tax and regulates subcellular localization of Tax.

    Science.gov (United States)

    Yamamoto, Keiyu; Ishida, Takaomi; Nakano, Kazumi; Yamagishi, Makoto; Yamochi, Tadanori; Tanaka, Yuetsu; Furukawa, Yoichi; Nakamura, Yusuke; Watanabe, Toshiki

    2011-01-01

    HTLV-1 Tax deregulates signal transduction pathways, transcription of genes, and cell cycle regulation of host cells, which is mainly mediated by its protein-protein interactions with host cellular factors. We previously reported an interaction of Tax with a histone methyltransferase (HMTase), SUV39H1. As the interaction was mediated by the SUV39H1 SET domain that is shared among HMTases, we examined the possibility of Tax interaction with another HMTase, SMYD3, which methylates histone H3 lysine 4 and activates transcription of genes, and studied the functional effects. Expression of endogenous SMYD3 in T cell lines and primary T cells was confirmed by immunoblotting analysis. Co-immuno-precipitaion assays and in vitro pull-down assay indicated interaction between Tax and SMYD3. The interaction was largely dependent on the C-terminal 180 amino acids of SMYD3, whereas the interacting domain of Tax was not clearly defined, although the N-terminal 108 amino acids were dispensable for the interaction. In the cotransfected cells, colocalization of Tax and SMYD3 was indicated in the cytoplasm or nuclei. Studies using mutants of Tax and SMYD3 suggested that SMYD3 dominates the subcellular localization of Tax. Reporter gene assays showed that nuclear factor-κB activation promoted by cytoplasmic Tax was enhanced by the presence of SMYD3, and attenuated by shRNA-mediated knockdown of SMYD3, suggesting an increased level of Tax localization in the cytoplasm by SMYD3. Our study revealed for the first time Tax-SMYD3 direct interaction, as well as apparent tethering of Tax by SMYD3, influencing the subcellular localization of Tax. Results suggested that SMYD3-mediated nucleocytoplasmic shuttling of Tax provides one base for the pleiotropic effects of Tax, which are mediated by the interaction of cellular proteins localized in the cytoplasm or nucleus. PMID:21054678

  16. Subcellular localisation of radionuclides by transmission electronic microscopy in aquatic and terrestrial organisms

    International Nuclear Information System (INIS)

    The global framework of this study is to go further in the understanding of the involved mechanisms of uranium and selenium internalisation at the subcellular level and of their toxicity towards several aquatic and terrestrial organisms. In this context, the applications and performances of a Scanning Transmission Electron Microscope (TEM/STEM) equipped with CCD camera and Energy-Dispersive- X-Ray (EDAX) analysis are reported. The principal merit of this equipment is the clear expression of element distribution with nanometer resolution. The sample for TEM analysis were prepared in ultrathin sections of 70-140 nm (thickness) and those for EDAX in sections of 200-500 nm. This method offers the possibility of a direct correlation between histological image and distribution map of trace elements. For each sample, following TEM analysis, EDAX spectra or EDAX mapping were also recorded to confirm the identity of the electron dense material in the scanned sections. Demonstration of the usefulness of this method to understand the bioaccumulation mechanisms and to study the effect of the pollutant uptake at the subcellular level was performed for target organs of a metal (U) and a metalloid (Se) in various biological models: a higher rooted plant (Phaseolus vulgaris)) and a freshwater invertebrate (Orconectes Limosus) and a unicellular green alga (Chlamydomonas reinhardtii)). TEM-EDAX analysis revealed the presence of U-deposits in gills and digestive gland in crayfish, and in vacuoles or in the cytoplasm of different rooted cells bean. In the alga, the accumulation of Se was found in electron-dense granules within cytoplasm associated with ultrastructural changes and starch accumulation. (author)

  17. Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

    Directory of Open Access Journals (Sweden)

    Amanda Getty

    Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

  18. Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

    Science.gov (United States)

    Schilling, Stephan; Lindner, Christiane; Koch, Birgit; Wermann, Michael; Rahfeld, Jens-Ulrich; von Bohlen, Alex; Rudolph, Thomas; Reuter, Gunter; Demuth, Hans-Ulrich

    2007-09-25

    Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes. PMID:17722885

  19. Prolactin kinase activity in bovine anterior pituitary sub-cellular fractions.

    Science.gov (United States)

    Wicks, J R; Brooks, C L

    1999-01-25

    Bovine anterior pituitary cells phosphorylate prolactin (PRL). We describe the phosphorylation of endogenous and exogenous bPRL in highly enriched subcellular fractions of bovine anterior pituitary using [gamma-32P]-ATP. 32P-labeling of endogenous and exogenous bPRL occurred in all subcellular membrane fractions, but most significantly in the fraction enriched for secretory granules. Zn2+ (0.8 mM), Cu2+ (0.8 mM), and Mn2+ (9.8 mM) increased bPRL phosphorylation by 268, 214, and 154%, respectively, relative to basal phosphorylation with no added cations. Neither Mg2+ (10 mM) nor Ca2+ (0.9 mM) increased bPRL phosphorylation above basal levels. Phosphorylation was dependent on the concentration of Zn2+ with an apparent Km of 570 microM. bPRL phosphorylation occurred over a wide pH range of 5.9-8.3, with the greatest activity at pH of 6.7 or greater. Phosphorylation of bPRL was time-dependent. The apparent Kms of the bPRL kinase for exogenous bPRL and ATP were 15.3 and 267 microM, respectively. bPRL incorporation of 32P was unaffected by the presence of calcium and calmodulin, cAMP, phosphotidylserine and diolein, or spermine. From these results we conclude that in vitro phosphorylation of bPRL occurs under physiological conditions that would be found in pituitary cells. PMID:10195699

  20. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    Science.gov (United States)

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  1. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Exposure to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mengjiao [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong, E-mail: wwang@ust.hk [State Key Laboratory in Marine Pollution, Section of Marine Ecology and Biotechnology, Division of Life Science, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)

    2011-01-25

    The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd{sup 2+}], 0.01-84 nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd{sup 2+}] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd{sup 2+}] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC{sub 2} being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs' detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd{sup 2+}] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.

  2. ESLpred2: improved method for predicting subcellular localization of eukaryotic proteins

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2008-11-01

    Full Text Available Abstract Background The expansion of raw protein sequence databases in the post genomic era and availability of fresh annotated sequences for major localizations particularly motivated us to introduce a new improved version of our previously forged eukaryotic subcellular localizations prediction method namely "ESLpred". Since, subcellular localization of a protein offers essential clues about its functioning, hence, availability of localization predictor would definitely aid and expedite the protein deciphering studies. However, robustness of a predictor is highly dependent on the superiority of dataset and extracted protein attributes; hence, it becomes imperative to improve the performance of presently available method using latest dataset and crucial input features. Results Here, we describe augmentation in the prediction performance obtained for our most popular ESLpred method using new crucial features as an input to Support Vector Machine (SVM. In addition, recently available, highly non-redundant dataset encompassing three kingdoms specific protein sequence sets; 1198 fungi sequences, 2597 from animal and 491 plant sequences were also included in the present study. First, using the evolutionary information in the form of profile composition along with whole and N-terminal sequence composition as an input feature vector of 440 dimensions, overall accuracies of 72.7, 75.8 and 74.5% were achieved respectively after five-fold cross-validation. Further, enhancement in performance was observed when similarity search based results were coupled with whole and N-terminal sequence composition along with profile composition by yielding overall accuracies of 75.9, 80.8, 76.6% respectively; best accuracies reported till date on the same datasets. Conclusion These results provide confidence about the reliability and accurate prediction of SVM modules generated in the present study using sequence and profile compositions along with similarity search

  3. Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Vrieling, E.G.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous c

  4. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  5. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs;

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthe...

  6. Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

    Directory of Open Access Journals (Sweden)

    Lewis Victoria

    2012-04-01

    Full Text Available Abstract Background Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres conformers of the normal cellular prion protein (PrPC, with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution. Findings Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication. Conclusions This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels.

  7. Spatial Variations in Archaeal Lipids of Surface Water and Core-Top Sediments in the South China Sea and Their Implications for Paleoclimate Studies▿†

    OpenAIRE

    Wei, Yuli; Wang, Jinxiang; Liu, Jie; Dong, Liang; Li, Li; Wang, Hui; Wang, Peng; Zhao, Meixun; Zhang, Chuanlun L.

    2011-01-01

    The South China Sea (SCS) is the largest marginal sea of the western Pacific Ocean, yet little is known about archaeal distributions and TEX86-based temperatures in this unique oceanic setting. Here we report findings of abundances in both core lipids (CL) and intact polar lipids (IPL) of Archaea from surface water (CL only) and core-top sediments from different regions of the SCS. TEX86-derived temperatures were also calculated for these samples. The surface water had extremely low abundance...

  8. Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.

    OpenAIRE

    Gohl, H P; Gröndahl, B; Thomm, M

    1995-01-01

    At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identifi...

  9. Diversity and Abundance of Ammonia-Oxidizing Archaeal Nitrite Reductase (nirK) Genes in Estuarine Sediments of San Francisco Bay

    Science.gov (United States)

    Reji, L.; Lee, J. A.; Damashek, J.; Francis, C. A.

    2013-12-01

    Nitrification, the microbially-mediated aerobic oxidation of ammonia to nitrate via nitrite, is an integral component of the global biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification, ammonia oxidation, is carried out by two distinct microbial groups: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Molecular ecological studies targeting the amoA gene have revealed the abundance and ubiquity of AOA in terrestrial as well as aquatic environments. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also encode a gene for copper-containing nitrite reductase (nirK). The distribution patterns and functional role of nirK in AOA remain mostly unknown; proposed functions include the indirect involvement in ammonia oxidation through the production of nitric oxide during nitrite reduction, and (2) nitrite detoxification. In the present study, the diversity and abundance of archaeal nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. In sediment samples collected from the San Francisco Bay estuary, two archaeal nirK variants (AnirKa and AnirKb) were amplified using specific primer sets. Overall, AnirKa was observed to be significantly more abundant than AnirKb in the sediment samples, with variation in relative abundance spanning two to three orders of magnitude between sampling sites. Phylogenetic analysis revealed a number of unique archaeal nirK sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, such as Nitrosopumilus maritimus. This study yielded new insights into the diversity and abundance of archaeal nirK genes in estuarine sediments, and highlights the importance of further investigating the physiological role of this gene in AOA, as well as its suitability as a marker gene for studying AOA in the environment.

  10. 古菌细胞膜脂在古菌群落组成及其对环境响应研究中的应用%Applications of archaeal membrane lipids in investigating archaeal community composition and its responses to environmental factors

    Institute of Scientific and Technical Information of China (English)

    曹鹏; 沈菊培; 贺纪正

    2012-01-01

    Archaea, as the third life form distinct from bacteria and eukaryota, widely distribute in various kinds of habitats, and play important roles in the biogeochemical cycles of carbon and nitrogen and in ecosystem functioning. As the biomarker of archaea, archaeal membrane lipids can be used to investigate the archaeal community composition and its responses to the environment. This paper introduced the structural characteristics of archaeal membrane lipids and the differences in the membrane lipids composition among different archaeal communities, and discussed the feasibility of using archeal membrane lipids in depicting archaeal community composition. The abundance of archaeal membrane lipids in the environment could be used to characterize the biomass of archaea, and the related results could complement and ascertain each other with the DNA-based bio-molecular approaches on the accuracy, analysis efficiency, and cost. Based on the description of the difficulties and importance of using archaeal membrane lipids to analyze the composition and abundance of archaeal communities, and by linking to the environmental factors such as temperature and pH that affected the archaeal community composition, the relationships between archaea and their habitats were further expatiated, and the evolution process of archaeal communities and its application prospects in the studies of geochemistry and geological events were analyzed.%古菌作为区别于细菌和真核生物的第3种生命形式广泛分布于各种生境,与碳、氮等元素的生物地球化学循环密切相关,在整个生态系统中具有重要作用.古菌细胞膜脂作为古菌重要的生物标志物,在其群落组成和对环境变化响应的研究中具有重要指示作用.本文介绍了古菌细胞膜脂的结构特征及不同古菌类群间细胞膜脂结构差异,用以表征古菌群落的组成特征.环境中细胞膜脂丰度可反映古菌生物量,并可与基于DNA的分子生物学

  11. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

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    Kunichika Tsumoto

    Full Text Available Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na+ channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na+ channels in the arrhythmogenicity under ischemia remain unclear.Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na+ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.We found in the myofiber model that the subcellular redistribution of the Na+ channels under myocardial ischemia, decreasing in Na+ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism did not reproduce the conduction slowing caused by the subcellular Na+ channel redistribution. Furthermore, Na+ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na+ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.Proarrhythmia of Na+ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na+ channel redistribution.

  12. Novel archaeal macrocyclic diether core membrane lipids in a methane-derived carbonate crust from a mud volcano in the Sorokin Trough, NE Black Sea

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    Alina Stadnitskaia

    2003-01-01

    Full Text Available A methane-derived carbonate crust was collected from the recently discovered NIOZ mud volcano in the Sorokin Trough, NE Black Sea during the 11th Training-through-Research cruise of the R/V Professor Logachev. Among several specific bacterial and archaeal membrane lipids present in this crust, two novel macrocyclic diphytanyl glycerol diethers, containing one or two cyclopentane rings, were detected. Their structures were tentatively identified based on the interpretation of mass spectra, comparison with previously reported mass spectral data, and a hydrogenation experiment. This macrocyclic type of archaeal core membrane diether lipid has so far been identified only in the deep-sea hydrothermal vent methanogen Methanococcus jannaschii. Here, we provide the first evidence that these macrocyclic diethers can also contain internal cyclopentane rings. The molecular structure of the novel diethers resembles that of dibiphytanyl tetraethers in which biphytane chains, containing one and two pentacyclic rings, also occur. Such tetraethers were abundant in the crust. Compound-specific isotope measurements revealed δ13C values of –104 to –111‰ for these new archaeal lipids, indicating that they are derived from methanotrophic archaea acting within anaerobic methane-oxidizing consortia, which subsequently induce authigenic carbonate formation.

  13. Identification and characterization of SNJ2, the first temperate pleolipovirus integrating into the genome of the SNJ1-lysogenic archaeal strain.

    Science.gov (United States)

    Liu, Ying; Wang, Jiao; Liu, Yang; Wang, Yuchen; Zhang, Ziqian; Oksanen, Hanna M; Bamford, Dennis H; Chen, Xiangdong

    2015-12-01

    Proviral regions have been identified in the genomes of many haloarchaea, but only a few archaeal halophilic temperate viruses have been studied. Here, we report a new virus, SNJ2, originating from archaeal strain Natrinema sp. J7-1. We demonstrate that this temperate virus coexists with SNJ1 virus and is dependent on SNJ1 for efficient production. Here, we show that SNJ1 is an icosahedral membrane-containing virus, whereas SNJ2 is a pleomorphic one. Instead of producing progeny virions and forming plaques, SNJ2 integrates into the host tRNA(Met) gene. The virion contains a discontinuous, circular, double-stranded DNA genome of 16 992 bp, in which both nicks and single-stranded regions are present preceded by a 'GCCCA' motif. Among 25 putative SNJ2 open reading frames (ORFs), five of them form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Furthermore, SNJ2-like proviruses containing the conserved gene cluster were identified in the chromosomes of archaea belonging to 10 different genera. Comparison of SNJ2 and these proviruses suggests that they employ a similar integration strategy into a tRNA gene. PMID:26331239

  14. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  15. Pyrosequencing reveals the influence of elevated atmospheric CO2 on the composition of archaeal communities in the rhizosphere of C3 and C4 crops

    Science.gov (United States)

    Nelson, D. M.; Cann, I. K.; Mackie, R. I.

    2008-12-01

    The projected increase in atmospheric CO2 concentrations throughout the 21st century is likely to increase aboveground and belowground plant productivity and cause changes in the quantity and quality of plant root exudates, although plants using C4 photosynthesis are likely to be only affected during times of drought (Leakey et al., 2006, Plant Physiology, 140, 779). Evidence is emerging from molecular tools that these changes may influence the abundance and composition of soil microbial communities that regulate key soil processes, such as nitrogen cycling (Lesaulnier et al., 2008, Environmental Microbiology, 10, 926). However, most molecular tools are not well-suited for comparing multiple samples at great sequencing depth, which is critical when considering soil microbial communities of high diversity. To overcome these limitations we used pyrosequencing and quantitative PCR (qPCR) of two genes (the V3 region of 16S rDNA and the amoA gene) to examine intra- and inter-treatment variability in the abundance and composition of microbial communities in the rhizosphere of soybean (C3) and maize (C4) grown in field conditions under ambient (~380 ppm) and elevated (~550 ppm) CO2 using FACE (free-air concentration enrichment) technology during the 2006 growing season in central Illinois. We specifically focused on archaeal communities because of their key role in nitrification (Leininger et al., 2006, Nature, 442, 806). The majority (>97%) of recovered sequences were from members of the phylum Crenarchaeota. Principle component analysis of sequence results from the V3 and amoA genes indicated significant (p<0.05) differences in the composition of rhizosphere archaeal communities between ambient and elevated CO2 beneath soybean, but not maize. qPCR suggested no significant difference in the abundance of archaea between treatments for soybean and maize. The lack of response of archaeal community composition beneath maize to elevated CO2 is consistent with relatively high

  16. Effect of supplementing coconut or krabok oil, rich in medium-chain fatty acids on ruminal fermentation, protozoa and archaeal population of bulls.

    Science.gov (United States)

    Panyakaew, P; Boon, N; Goel, G; Yuangklang, C; Schonewille, J Th; Hendriks, W H; Fievez, V

    2013-12-01

    Medium-chain fatty acids (MCFA), for example, capric acid (C10:0), myristic (C14:0) and lauric (C12:0) acid, have been suggested to decrease rumen archaeal abundance and protozoal numbers. This study aimed to compare the effect of MCFA, either supplied through krabok (KO) or coconut (CO) oil, on rumen fermentation, protozoal counts and archaeal abundance, as well as their diversity and functional organization. KO contains similar amounts of C12:0 as CO (420 and 458 g/kg FA, respectively), but has a higher proportion of C14:0 (464 v. 205 g/kg FA, respectively). Treatments contained 35 g supplemental fat per kg DM: a control diet with tallow (T); a diet with supplemental CO; and a diet with supplemental KO. A 4th treatment consisted of a diet with similar amounts of MCFA (i.e. C10:0+C12:0+C14:0) from CO and KO. To ensure isolipidic diets, extra tallow was supplied in the latter treatment (KO+T). Eight fistulated bulls (two bulls per treatment), fed a total mixed ration predominantly based on cassava chips, rice straw, tomato pomace, rice bran and soybean meal (1.5% of BW), were used. Both KO and CO increased the rumen volatile fatty acids, in particular propionate and decreased acetate proportions. Protozoal numbers were reduced through the supplementation of an MCFA source (CO, KO and KO+T), with the strongest reduction by KO. Quantitative real-time polymerase chain reaction assays based on archaeal primers showed a decrease in abundance of Archaea when supplementing with KO and KO+T compared with T and CO. The denaturing gradient gel electrophoresis profiles of the rumen archaeal population did not result in a grouping of treatments. Richness indices were calculated from the number of DGGE bands, whereas community organization was assessed from the Pareto-Lorenz evenness curves on the basis of DGGE band intensities. KO supplementation (KO and KO+T treatments) increased richness and evenness within the archaeal community. Further research including methane

  17. Impacts of temperature and pH on the distribution of archaeal lipids in Yunnan hot springs, China.

    Science.gov (United States)

    Wu, Weiyan; Zhang, Chuanlun L; Wang, Huanye; He, Liu; Li, Wenjun; Dong, Hailiang

    2013-01-01

    In culture experiments and many low temperature environments, the distribution of isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) commonly shows a strong correlation with temperature; however, this is often not the case in hot springs. We studied 26 hot springs in Yunnan, China, in order to determine whether temperature or other factors control the distribution of GDGTs in these environments. The hot springs ranged in temperature from 39.0 to 94.0°C, and in pH from 2.35 to 9.11. Water chemistry including nitrogen-, sulfur-, and iron species was also determined. Lipids from the samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Distributions of GDGTs in these hot springs were examined using cluster analysis, which resulted in two major groups. Group 1 was characterized by the lack of dominance of any individual GDGTs, while Group 2 was defined by the dominance of GDGT-0 or thaumarchaeol. Temperature was the main control on GDGT distribution in Group 1, whereas pH played an important role in the distribution of GDGTs in Group 2. However, no correlations were found between the distribution of GDGTs and any of the nitrogen-, sulfur-, or iron species. Results of this study indicate the dominance of temperature or pH control on archaeal lipid distribution, which can be better evaluated in the context of lipid classification.

  18. Impacts of temperature and pH on the distribution of archaeal lipids in Yunnan hot springs, China

    Directory of Open Access Journals (Sweden)

    Weiyan eWu

    2013-10-01

    Full Text Available In culture experiments and many low temperature environments, the distribution of isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs commonly shows a strong correlation with temperature; however, this is often not the case in hot springs. We studied 26 hot springs in Yunnan, China, in order to determine whether temperature or other factors control the distribution of GDGTs in these environments. The hot springs ranged in temperature from 39°C to 94°C, and in pH from 2.35 to 9.11. Water chemistry including nitrogen-, sulfur- and iron species was also determined. Lipids from the samples were analyzed using LC-MS (liquid chromatography-mass spectrometry. Distributions of GDGTs in these hot springs were examined using cluster analysis, which resulted in two major groups. Group 1 was characterized by the lack of dominance of any individual GDGTs, while Group 2 was defined by the dominance of GDGT-0 or thaumarchaeol. Temperature was the main control on GDGT distribution in Group 1, whereas pH played an important role in the distribution of GDGTs in Group 2. However, no correlations were found between the distribution of GDGTs and any of the nitrogen-, sulfur- or iron species. Results of this study indicate the predominance of temperature or pH control on archaeal lipid distribution, which can be better evaluated in the context of lipid classification.

  19. S-layers at second glance? Altiarchaeal grappling hooks (hami resemble archaeal S-layer proteins in structure and sequence

    Directory of Open Access Journals (Sweden)

    Alexandra Kristin Perras

    2015-06-01

    Full Text Available The uncultivated Ca. Altiarchaeum hamiconexum (formerly known as SM1 Euryarchaeon carries highly specialized nano-grappling hooks (hami on its cell surface. Until now little is known about the major protein forming these structured fibrous cell surface appendages, the genes involved or membrane anchoring of these filaments. These aspects were analyzed in depth in this study using environmental transcriptomics combined with imaging methods. Since a laboratory culture of this archaeon is not yet available, natural biofilm samples with high Ca. A. hamiconexum abundance were used for the entire analyses. The filamentous surface appendages spanned both membranes of the cell, which are composed of glycosyl-archaeol. The hami consisted of multiple copies of the same protein, the corresponding gene of which was identified via metagenome-mapped transcriptome analysis. The hamus subunit proteins, which are likely to self-assemble due to their predicted beta sheet topology, revealed no similiarity to known microbial flagella-, archaella-, fimbriae- or pili-proteins, but a high similarity to known S-layer proteins of the archaeal phylum at their N-terminal region (47-44% identity. Our results provide new insights into the structure of the unique hami and their major protein and indicate their divergent evolution with S-layer proteins.

  20. Archaeal Diversity in Biofilm Technologies Applied to Treat Urban and Industrial Wastewater: Recent Advances and Future Prospects

    Directory of Open Access Journals (Sweden)

    Jesús González-López

    2013-09-01

    Full Text Available Biological wastewater treatment (WWT frequently relies on biofilms for the removal of anthropogenic contaminants. The use of inert carrier materials to support biofilm development is often required, although under certain operating conditions microorganisms yield structures called granules, dense aggregates of self-immobilized cells with the characteristics of biofilms maintained in suspension. Molecular techniques have been successfully applied in recent years to identify the prokaryotic communities inhabiting biofilms in WWT plants. Although methanogenic Archaea are widely acknowledged as key players for the degradation of organic matter in anaerobic bioreactors, other biotechnological functions fulfilled by Archaea are less explored, and research on their significance and potential for WWT is largely needed. In addition, the occurrence of biofilms in WWT plants can sometimes be a source of operational problems. This is the case for membrane bioreactors (MBR, an advanced technology that combines conventional biological treatment with membrane filtration, which is strongly limited by biofouling, defined as the undesirable accumulation of microbial biofilms and other materials on membrane surfaces. The prevalence and spatial distribution of archaeal communities in biofilm-based WWT as well as their role in biofouling are reviewed here, in order to illustrate the significance of this prokaryotic cellular lineage in engineered environments devoted to WWT.

  1. Human settlement as driver of bacterial, but not of archaeal, ammonia oxidizers abundance and community structure in tropical stream sediments

    Directory of Open Access Journals (Sweden)

    Mariana De Paula Reis

    2015-08-01

    Full Text Available Ammonia-oxidizing archaea (AOA and bacteria (AOB are a diverse and functionally important group in the nitrogen cycle. Nevertheless, AOA and AOB communities driving this process remain uncharacterized in tropical freshwater sediment. Here, the effect of human settlement on the AOA and AOB diversity and abundance have been assessed by phylogenetic and quantitative PCR analyses, using archaeal and bacterial amoA and 16S rRNA genes. Overall, each environment contained specific clades of amoA and 16S rRNA genes sequences, suggesting that selective pressures lead to AOA and AOB inhabiting distinct ecological niches. Human settlement activities, as derived from increased metal and mineral nitrogen contents, appear to cause a response among the AOB community, with Nitrosomonas taking advantage over Nitrosospira in impacted environments. We also observed a dominance of AOB over AOA in mining-impacted sediments, suggesting that AOB might be the primary drivers of ammonia oxidation in these sediments. In addition, ammonia concentrations demonstrated to be the driver for the abundance of AOA, with an inversely proportional correlation between them. Our findings also revealed the presence of novel ecotypes of Thaumarchaeota, such as those related to the obligate acidophilic Nitrosotalea devanaterra at ammonia-rich places of circumneutral pH. These data add significant new information regarding AOA and AOB from tropical freshwater sediments, albeit future studies would be required to provide additional insights into the niche differentiation among these microorganisms.

  2. Temporal and Spatial Coexistence of Archaeal and Bacterial amoA Genes and Gene Transcripts in Lake Lucerne

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    Elisabeth W. Vissers

    2013-01-01

    Full Text Available Despite their crucial role in the nitrogen cycle, freshwater ecosystems are relatively rarely studied for active ammonia oxidizers (AO. This study of Lake Lucerne determined the abundance of both amoA genes and gene transcripts of ammonia-oxidizing archaea (AOA and bacteria (AOB over a period of 16 months, shedding more light on the role of both AO in a deep, alpine lake environment. At the surface, at 42 m water depth, and in the water layer immediately above the sediment, AOA generally outnumbered AOB. However, in the surface water during summer stratification, when both AO were low in abundance, AOB were more numerous than AOA. Temporal distribution patterns of AOA and AOB were comparable. Higher abundances of amoA gene transcripts were observed at the onset and end of summer stratification. In summer, archaeal amoA genes and transcripts correlated negatively with temperature and conductivity. Concentrations of ammonium and oxygen did not vary enough to explain the amoA gene and transcript dynamics. The observed herbivorous zooplankton may have caused a hidden flux of mineralized ammonium and a change in abundance of genes and transcripts. At the surface, AO might have been repressed during summer stratification due to nutrient limitation caused by active phytoplankton.

  3. High Oxygen Concentration Increases the Abundance and Activity of Bacterial Rather than Archaeal Nitrifiers in Rice Field Soil.

    Science.gov (United States)

    Ke, Xiubin; Lu, Wei; Conrad, Ralf

    2015-11-01

    Oxygen is considered as a limiting factor for nitrification in rice paddy soil. However, little is known about how the nitrifying microbial community responds to different oxygen concentrations at community and transcript level. In this study, soil and roots were harvested from 50-day-old rice microcosms and were incubated for up to 45 days under two oxygen concentrations: 2 % O(2) and 20 % O(2) (ambient air). Nitrification rates were measured from the accumulation of nitrite plus nitrate. The population dynamics of bacterial (AOB) and archaeal (AOA) ammonia oxidizers was determined from the abundance (using quantitative PCR (qPCR)) and composition (using terminal restriction fragment length polymorphism and cloning/sequencing) of their amoA genes, that of nitrite oxidizers (NOB) by quantifying the nxrA gene of Nitrobacter spp. and the 16S rRNA gene of Nitrospira spp. The activity of the nitrifiers was determined by quantifying the copy numbers of amoA and nxrA transcripts (using RT-qPCR). Different oxygen concentrations did not affect the community compositions of AOB, AOA, and NOB, which however were different between surface soil, bottom soil, and rice roots. However, nitrification rates were higher under ambient air than 2 % O(2), and abundance and transcript activities of AOB, but not of AOA, were also higher. Abundance and transcript copy numbers of Nitrobacter were also higher at ambient air. These results indicate that AOB and NOB, but not AOA, were sensitive to oxygen availability. PMID:26054702

  4. Quantification of bacterial and archaeal symbionts in high and low microbial abundance sponges using real-time PCR

    KAUST Repository

    Bayer, Kristina

    2014-07-09

    In spite of considerable insights into the microbial diversity of marine sponges, quantitative information on microbial abundances and community composition remains scarce. Here, we established qPCR assays for the specific quantification of four bacterial phyla of representative sponge symbionts as well as the kingdoms Eubacteria and Archaea. We could show that the 16S rRNA gene numbers of Archaea, Chloroflexi, and the candidate phylum Poribacteria were 4-6 orders of magnitude higher in high microbial abundance (HMA) than in low microbial abundance (LMA) sponges and that actinobacterial 16S rRNA gene numbers were 1-2 orders higher in HMA over LMA sponges, while those for Cyanobacteria were stable between HMA and LMA sponges. Fluorescence in situ hybridization of Aplysina aerophoba tissue sections confirmed the numerical dominance of Chloroflexi, which was followed by Poribacteria. Archaeal and actinobacterial cells were detected in much lower numbers. By use of fluorescence-activated cell sorting as a primer- and probe-independent approach, the dominance of Chloroflexi, Proteobacteria, and Poribacteria in A. aerophoba was confirmed. Our study provides new quantitative insights into the microbiology of sponges and contributes to a better understanding of the HMA/LMA dichotomy. The authors quantified sponge symbionts in eight sponge species from three different locations by real time PCR targetting 16S rRNA genes. Additionally, FISH was performed and diversity and abundance of singularized microbial symbionts from Aplysina aerophoba was determined for a comprehensive quantification work. © 2014 Federation of European Microbiological Societies.

  5. Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient

    International Nuclear Information System (INIS)

    Chronic exposure to polluted field conditions can impact metal bioavailability in prey and may influence metal transfer to predators. The present study investigated the assimilation of Cd, Hg and organic carbon by grass shrimp Palaemonetes pugio, collected along an impact gradient within the New York/New Jersey Harbor Estuary. Adult shrimp were collected from five Staten Island, New York study sites, fed 109Cd- or 203Hg-labeled amphipods or 14C-labeled meals and analyzed for assimilation efficiencies (AE). Subsamples of amphipods and shrimp were subjected to subcellular fractionation to isolate metal associated with a compartment presumed to contain trophically available metal (TAM) (metal associated with heat-stable proteins [HSP - e.g., metallothionein-like proteins], heat-denatured proteins [HDP - e.g., enzymes] and organelles [ORG]). TAM-109Cd% and TAM-203Hg% in radiolabeled amphipods were ∼64% and ∼73%, respectively. Gradients in AE-109Cd% (∼54% to ∼75%) and AE-203Hg% (∼61% to ∼78%) were observed for grass shrimp, with the highest values exhibited by shrimp collected from sites within the heavily polluted Arthur Kill complex. Population differences in AE-14C% were not observed. Assimilated 109Cd% partitioned to the TAM compartment in grass shrimp varied between ∼67% and ∼75%. 109Cd bound to HSP in shrimp varied between ∼15% and ∼47%, while 109Cd associated with metal-sensitive HDP was ∼17% to ∼44%. Percentages of assimilated 109Cd bound to ORG were constant at ∼10%. Assimilated 203Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated 203Hg bound to HDP (∼47%) and ORG (∼11%) did not vary among populations and partitioning of 203Hg to HSP was not observed. Using a simplified biokinetic model of metal accumulation from the diet, it is estimated that site-specific variability in Cd AE by shrimp and tissue Cd burdens in field-collected prey (polychaetes Nereis spp.) could potentially

  6. Movies of cellular and sub-cellular motion by digital holographic microscopy

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    Yu Lingfeng

    2006-03-01

    Full Text Available Abstract Background Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. Methods A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Results Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable

  7. Subcellular adaptation of the human diaphragm in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Orozco-Levi, M; Gea, J; Lloreta, J L; Félez, M; Minguella, J; Serrano, S; Broquetas, J M

    1999-02-01

    Pulmonary hyperinflation impairs the function of the diaphragm in patients with chronic obstructive pulmonary disease (COPD). However, it has been recently demonstrated that the muscle can counterbalance this deleterious effect, remodelling its structure (i.e. changing the proportion of different types of fibres). The aim of this study was to investigate whether the functional impairment present in COPD patients can be associated with structural subcellular changes of the diaphragm. Twenty individuals (60+/-9 yrs, 11 COPD patients and 9 subjects with normal spirometry) undergoing thoracotomy were included. Nutritional status and respiratory function were evaluated prior to surgery. Then, small samples of the costal diaphragm were obtained and processed for electron microscopy analysis. COPD patients showed a mean forced expiratory volume in one second (FEV1) of 60+/-9% predicted, a higher concentration of mitochondria (n(mit)) in their diaphragm than controls (0.62+/-0.16 versus 0.46+/-0.16 mitochondrial transections (mt) x microm(-2), p37%) disclosed not only a higher n(mit) (0.63+/-0.17 versus 0.43+/-0.07 mt x microm(-2), p<0.05) but shorter sarcomeres (L(sar)) than subjects without this functional abnormality (2.08+/-0.16 to 2.27+/-0.15 microm, p<0.05). Glycogen stores were similar in COPD and controls. The severity of airways obstruction (i.e. FEV1) was associated with n(mit) (r=-0.555, p=0.01), while the amount of air trapping (i.e. RV/TLC) was found to correlate with both n(mit) (r=0.631, p=0.005) and L(sar) (r=-0.526, p<0.05). Finally, maximal inspiratory pressure (PI,max) inversely correlated with n(mit) (r=-0.547, p=0.01). In conclusion, impairment in lung function occurring in patients with chronic obstructive pulmonary disease is associated with subcellular changes in their diaphragm, namely a shortening in the length of sarcomeres and an increase in the concentration of mitochondria. These changes form a part of muscle remodelling, probably contributing

  8. Subcellular localization of total and activated Src kinase in African American and Caucasian breast cancer.

    Directory of Open Access Journals (Sweden)

    Muralidharan Anbalagan

    Full Text Available BACKGROUND: Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups. METHODOLOGY/PRINCIPAL FINDINGS: The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC and ERα positive breast cancer (ER+BC, cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007-2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns. Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P<0.0001 in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028; conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106. Comparison between African American (n = 21 and Caucasian ER+BC (n = 16 revealed no significant difference in expression and localization of total Src and p-Y416Src. TNBC cases positive for lymph node metastasis showed elevated membrane p-Y416Src compared to lymph node negative TNBC (P = 0.027. CONCLUSION/SIGNIFICANCE: Total Src and p-Y416Src were expressed higher in cancer compared to adjacent normal ducts. Cytoplasmic total Src and membrane p-Y416Src were

  9. Assimilation and subcellular partitioning of elements by grass shrimp collected along an impact gradient

    Energy Technology Data Exchange (ETDEWEB)

    Seebaugh, David R. [Department of Biology, Graduate School and University Center, City University of New York, 365 Fifth Avenue, New York, NY 10016 (United States); Wallace, William G., E-mail: wallace@mail.csi.cuny.edu [Department of Biology, Graduate School and University Center, City University of New York, 365 Fifth Avenue, New York, NY 10016 (United States); Department of Biology, College of Staten Island, 6S-310, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States)

    2009-06-28

    Chronic exposure to polluted field conditions can impact metal bioavailability in prey and may influence metal transfer to predators. The present study investigated the assimilation of Cd, Hg and organic carbon by grass shrimp Palaemonetes pugio, collected along an impact gradient within the New York/New Jersey Harbor Estuary. Adult shrimp were collected from five Staten Island, New York study sites, fed {sup 109}Cd- or {sup 203}Hg-labeled amphipods or {sup 14}C-labeled meals and analyzed for assimilation efficiencies (AE). Subsamples of amphipods and shrimp were subjected to subcellular fractionation to isolate metal associated with a compartment presumed to contain trophically available metal (TAM) (metal associated with heat-stable proteins [HSP - e.g., metallothionein-like proteins], heat-denatured proteins [HDP - e.g., enzymes] and organelles [ORG]). TAM-{sup 109}Cd% and TAM-{sup 203}Hg% in radiolabeled amphipods were {approx}64% and {approx}73%, respectively. Gradients in AE-{sup 109}Cd% ({approx}54% to {approx}75%) and AE-{sup 203}Hg% ({approx}61% to {approx}78%) were observed for grass shrimp, with the highest values exhibited by shrimp collected from sites within the heavily polluted Arthur Kill complex. Population differences in AE-{sup 14}C% were not observed. Assimilated {sup 109}Cd% partitioned to the TAM compartment in grass shrimp varied between {approx}67% and {approx}75%. {sup 109}Cd bound to HSP in shrimp varied between {approx}15% and {approx}47%, while {sup 109}Cd associated with metal-sensitive HDP was {approx}17% to {approx}44%. Percentages of assimilated {sup 109}Cd bound to ORG were constant at {approx}10%. Assimilated {sup 203}Hg% associated with TAM in grass shrimp did not exhibit significant variation. Percentages of assimilated {sup 203}Hg bound to HDP ({approx}47%) and ORG ({approx}11%) did not vary among populations and partitioning of {sup 203}Hg to HSP was not observed. Using a simplified biokinetic model of metal accumulation from

  10. Protein subcellular localization prediction based on compartment-specific features and structure conservation

    Directory of Open Access Journals (Sweden)

    Lo Allan

    2007-09-01

    Full Text Available Abstract Background Protein subcellular localization is crucial for genome annotation, protein function prediction, and drug discovery. Determination of subcellular localization using experimental approaches is time-consuming; thus, computational approaches become highly desirable. Extensive studies of localization prediction have led to the development of several methods including composition-based and homology-based methods. However, their performance might be significantly degraded if homologous sequences are not detected. Moreover, methods that integrate various features could suffer from the problem of low coverage in high-throughput proteomic analyses due to the lack of information to characterize unknown proteins. Results We propose a hybrid prediction method for Gram-negative bacteria that combines a one-versus-one support vector machines (SVM model and a structural homology approach. The SVM model comprises a number of binary classifiers, in which biological features derived from Gram-negative bacteria translocation pathways are incorporated. In the structural homology approach, we employ secondary structure alignment for structural similarity comparison and assign the known localization of the top-ranked protein as the predicted localization of a query protein. The hybrid method achieves overall accuracy of 93.7% and 93.2% using ten-fold cross-validation on the benchmark data sets. In the assessment of the evaluation data sets, our method also attains accurate prediction accuracy of 84.0%, especially when testing on sequences with a low level of homology to the training data. A three-way data split procedure is also incorporated to prevent overestimation of the predictive performance. In addition, we show that the prediction accuracy should be approximately 85% for non-redundant data sets of sequence identity less than 30%. Conclusion Our results demonstrate that biological features derived from Gram-negative bacteria translocation

  11. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    Science.gov (United States)

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  12. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types

    International Nuclear Information System (INIS)

    Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells. - Selenium assimilation by a predatory aquatic insect depends on Se availability in the cells of its prey

  13. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    International Nuclear Information System (INIS)

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the location of Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments

  14. Protein subcellular localization in human and hamster cell lines: employing local ternary patterns of fluorescence microscopy images.

    Science.gov (United States)

    Tahir, Muhammad; Khan, Asifullah; Kaya, Hüseyin

    2014-01-01

    Discriminative feature extraction technique is always required for the development of accurate and efficient prediction systems for protein subcellular localization so that effective drugs can be developed. In this work, we showed that Local Ternary Patterns (LTPs) effectively exploit small variations in pixel intensities; present in fluorescence microscopy based protein images of human and hamster cell lines. Further, Synthetic Minority Oversampling Technique is applied to balance the feature space for the classification stage. We observed that LTPs coupled with data balancing technique could enable a classifier, in this case support vector machine, to yield good performance. The proposed ensemble based prediction system, using 10-fold cross-validation, has yielded better performance compared to existing techniques in predicting various subcellular compartments for both 2D HeLa and CHO datasets. The proposed predictor is available online at: http://111.68.99.218/Protein_SubLoc/, which is freely accessible to the public. PMID:23988793

  15. Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

    Science.gov (United States)

    Pratt, Evan P S; Owens, Jake L; Hockerman, Gregory H; Hu, Chang-Deng

    2016-01-01

    Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software. PMID:27515079

  16. Perspective: On the importance of hydrodynamic interactions in the subcellular dynamics of macromolecules

    Science.gov (United States)

    Skolnick, Jeffrey

    2016-09-01

    An outstanding challenge in computational biophysics is the simulation of a living cell at molecular detail. Over the past several years, using Stokesian dynamics, progress has been made in simulating coarse grained molecular models of the cytoplasm. Since macromolecules comprise 20%-40% of the volume of a cell, one would expect that steric interactions dominate macromolecular diffusion. However, the reduction in cellular diffusion rates relative to infinite dilution is due, roughly equally, to steric and hydrodynamic interactions, HI, with nonspecific attractive interactions likely playing rather a minor role. HI not only serve to slow down long time diffusion rates but also cause a considerable reduction in the magnitude of the short time diffusion coefficient relative to that at infinite dilution. More importantly, the long range contribution of the Rotne-Prager-Yamakawa diffusion tensor results in temporal and spatial correlations that persist up to microseconds and for intermolecular distances on the order of protein radii. While HI slow down the bimolecular association rate in the early stages of lipid bilayer formation, they accelerate the rate of large scale assembly of lipid aggregates. This is suggestive of an important role for HI in the self-assembly kinetics of large macromolecular complexes such as tubulin. Since HI are important, questions as to whether continuum models of HI are adequate as well as improved simulation methodologies that will make simulations of more complex cellular processes practical need to be addressed. Nevertheless, the stage is set for the molecular simulations of ever more complex subcellular processes.

  17. Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

    Science.gov (United States)

    Surmacz, L; Wiejak, J; Wyroba, E

    2001-01-01

    SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

  18. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.

    Science.gov (United States)

    Baghirova, Sabina; Hughes, Bryan G; Hendzel, Michael J; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C. PMID:26740924

  19. Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

    Institute of Scientific and Technical Information of China (English)

    邱全胜; 王泽宙; 蔡起贵; 姜荣锡

    2002-01-01

    The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by microprojectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclohexylcarbodiimid, indicating that the cytoplasmic DHN1 was newly synthesized under osmotic stress. Furthermore, ABA promoted the presence of green fluorescence in the cytoplasm, and the GFP fluorescence was visualized within a shorter time under a higher osmotic potential.

  20. ELECTRON MICROSCOPIC AUTORADIOGRAPHIC STUDY ON SUBCELLULAR LOCALIZATION OF FISSION PRODUCT 147Pm IN TISSUE CELLS

    Institute of Scientific and Technical Information of China (English)

    朱寿彭; 汪源长

    1994-01-01

    The early risk of internal contaminated accumualtion of 147Pm is in blood cells and endothelial cells,especially in red blood cells.Then 147Pm is selectively deposited in ultrastructure of liver cells,such as in nucleus,nucleolus,rough endoplasmic reticulum,mitochondria and microbodies,Dense tracks also appear in mitochondria and lysosome of pedal cells within renal corpuscle,and so dose in nucleus as well as in mitochondria and microbodies of epicyte of kidney near-convoluted tubule.With the prolongation of observing time,147Pm is selectively and steadily depostied in subcellular level of organic ocmponent for bone.Substantial amount of 147Pm is taken up into the nuclear fraction of osteoclasts and osteoblasts.Particularly,in organelles 147Pm is mainly accumulated in rough endoplasmic reticulum and in mitochondria.Autoradiographic tracks especially localize in combined point between Golgi complex and transitive vesicle of rough endoplasmic reticulum.In addition,numerous 147Pm deposited in collagenous fibre within interstitial of bone cells is hardly excreted.

  1. A General Procedure to Study Subcellular Models of Transsynaptic Signaling at Inhibitory Synapses.

    Science.gov (United States)

    Lupascu, Carmen A; Morabito, Annunziato; Merenda, Elisabetta; Marinelli, Silvia; Marchetti, Cristina; Migliore, Rosanna; Cherubini, Enrico; Migliore, Michele

    2016-01-01

    Computational modeling of brain circuits requires the definition of many parameters that are difficult to determine from experimental findings. One way to help interpret these data is to fit them using a particular kinetic model. In this paper, we propose a general procedure to fit individual synaptic events recorded from voltage clamp experiments. Starting from any given model description (mod file) in the NEURON simulation environment, the procedure exploits user-defined constraints, dependencies, and rules for the parameters of the model to fit the time course of individual spontaneous synaptic events that are recorded experimentally. The procedure, implemented in NEURON, is currently available in ModelDB. A Python version is installed, and will be soon available for public use, as a standalone task in the Collaboratory Portal of the Human Brain Project. To illustrate the potential application of the procedure, we tested its use with various sets of experimental data on GABAergic synapses; gephyrin and gephyrin-dependent pathways were chosen as a suitable example of a kinetic model of synaptic transmission. For individual spontaneous inhibitory events in hippocampal pyramidal CA1 neurons, we found that gephyrin-dependent subcellular pathways may shape synaptic events at different levels, and can be correlated with cell- or event-specific activity history and/or pathological conditions. PMID:27445784

  2. Subcellular distribution of Al, Cu, Mg, Mn and other elements in the human liver

    Energy Technology Data Exchange (ETDEWEB)

    Chen, C.; Zhang Peiqun; Lu Xiangli; Hou Xiaolin; Chai Zhifang [Institute of High Energy Physics and Laboratory of Nuclear Analysis Techniques, Chinese Academy of Sciences, Beijing (China)

    1999-03-01

    The elemental concentrations and chemical species of Al, Br, Cl, Cu, K, Mg, Mn, Na and I in human liver and its subcellular fractions were studied by several biochemical techniques combined with neutron activation analysis. The highest concentrations of Al, Mg, and I were found in the nuclei, whereas those of Br, Cu, Cl, K, Mn and Na in the cytosol. About 20% of Br, half of Al and most of Cu (78.8%), Mg (65.9%) and Mn (80.6%) remained in the cellulose bags after dialysis of liver homogenate, which were suggested to be bound to macromolecules. K (100%) and more than 95% of Cl and Na were found to be in the dialyzates. Similar results were found in the fractions of nuclei, mitochondria, lysosome and microsome, respectively, after the same treatment. Further study was carried out to elucidate the elemental distribution in the cytosol by ethanol precipitation and by ammonium sulfate fractionation. The results suggested that several kinds of Cu-, Mn- and Mg-bound proteins existed in the cytosol of human liver cells. (orig.) With 2 figs., 6 tabs., 14 refs.

  3. Protein Subcellular Localization Prediction and Genomic Polymorphism Analysis of the SARS Coronavirus

    Institute of Scientific and Technical Information of China (English)

    季星来; 柳树群; 李岭; 孙之荣

    2004-01-01

    The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus (CoV).Several sequences of the complete genome of SARS-CoV have been determined.The subcellular localization (SubLocation) of annotated open-reading frames of the SARS-CoV genome was predicted using a support vector machine.Several gene products were predicted to locate in the Golgi body and cell nucleus.The SubLocation information was combined with predicted transmembrane information to develop a model of the viral life cycle.The results show that this information can be used to predict the functions of genes and even the virus pathogenesis.In addition,the entire SARS viral genome sequences currently available in GenBank were compared to identify the sequence variations among different isolates.Some variations in the Hong Kong strains may be related to the special clinical manifestations and provide clues for understanding the relationship between gene functions and evolution.These variations reflect the evolution of the SARS virus in human populations and may help development of a vaccine.

  4. Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

    Science.gov (United States)

    Di Sansebastiano, Gian Pietro; Rizzello, Francesca; Durante, Miriana; Caretto, Sofia; Nisi, Rossella; De Paolis, Angelo; Faraco, Marianna; Montefusco, Anna; Piro, Gabriella; Mita, Giovanni

    2015-05-20

    Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

  5. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Bindesh Shrestha

    Full Text Available Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI, an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen, were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  6. Dynamic full field OCT: metabolic contrast at subcellular level (Conference Presentation)

    Science.gov (United States)

    Apelian, Clement; Harms, Fabrice; Thouvenin, Olivier; Boccara, Claude A.

    2016-03-01

    Cells shape or density is an important marker of tissues pathology. However, individual cells are difficult to observe in thick tissues frequently presenting highly scattering structures such as collagen fibers. Endogenous techniques struggle to image cells in these conditions. Moreover, exogenous contrast agents like dyes, fluorophores or nanoparticles cannot always be used, especially if non-invasive imaging is required. Scatterers motion happening down to the millisecond scale, much faster than the still and highly scattering structures (global motion of the tissue), allowed us to develop a new approach based on the time dependence of the FF-OCT signals. This method reveals hidden cells after a spatiotemporal analysis based on singular value decomposition and wavelet analysis concepts. It does also give us access to local dynamics of imaged scatterers. This dynamic information is linked with the local metabolic activity that drives these scatterers. Our technique can explore subcellular scales with micrometric resolution and dynamics ranging from the millisecond to seconds. By this mean we studied a wide range of tissues, animal and human in both normal and pathological conditions (cancer, ischemia, osmotic shock…) in different organs such as liver, kidney, and brain among others. Different cells, undetectable with FF-OCT, were identified (erythrocytes, hepatocytes…). Different scatterers clusters express different characteristic times and thus can be related to different mechanisms that we identify with metabolic functions. We are confident that the D-FFOCT, by accessing to a new spatiotemporal metabolic contrast, will be a leading technique on tissue imaging and for better medical diagnosis.

  7. Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

    Science.gov (United States)

    Chandra, Subhash; Morrison, George H.

    1995-05-01

    The transport of K+, Na+, and Ca2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 [mu]m spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged (13C, 15N) amino acid, -arginine. The labeled amino acid was imaged at mass 28 with a Cs+ primary ion beam as the 28(13C15N)- species. After a 4 h exposure of LLC-PK1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.

  8. Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

    Energy Technology Data Exchange (ETDEWEB)

    Stekhoven, Daniel J. [Univ. of Zurich (Switzerland); Omasits, Ulrich [Univ. of Zurich (Switzerland); ETH Zurich (Switzerland); Quebatte, Maxime [Univ. of Basel (Switzerland); Dehio, Christoph [Univ. of Basel (Switzerland); Ahrens, Christian H. [Univ. of Zurich (Switzerland)

    2014-03-01

    Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled us to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.

  9. Microscopy with spatial filtering for sorting particles and monitoring subcellular morphology

    Science.gov (United States)

    Zheng, Jing-Yi; Qian, Zhen; Pasternack, Robert M.; Boustany, Nada N.

    2009-02-01

    Optical scatter imaging (OSI) was developed to non-invasively track real-time changes in particle morphology with submicron sensitivity in situ without exogenous labeling, cell fixing, or organelle isolation. For spherical particles, the intensity ratio of wide-to-narrow angle scatter (OSIR, Optical Scatter Image Ratio) was shown to decrease monotonically with diameter and agree with Mie theory. In living cells, we recently reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl-xL transmembrane domain, and could not be observed by fluorescence or differential interference contrast images. Here we further extend the ability of morphology assessment by adopting a digital micromirror device (DMD) for Fourier filtering. When placed in the Fourier plane the DMD can be used to select scattering intensities at desired combination of scattering angles. We designed an optical filter bank consisting of Gabor-like filters with various scales and rotations based on Gabor filters, which have been widely used for localization of spatial and frequency information in digital images and texture analysis. Using a model system consisting of mixtures of polystyrene spheres and bacteria, we show how this system can be used to sort particles on a microscopic slide based on their size, orientation and aspect ratio. We are currently applying this technique to characterize the morphology of subcellular organelles to help understand fundamental biological processes.

  10. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants.

    Science.gov (United States)

    Herrera, Rito; Álvarez, María C; Gelis, Samuel; Ramos, José

    2013-09-15

    Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

  11. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  12. Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

    Science.gov (United States)

    Pierret, P; Vallée, A; Mechawar, N; Dower, N A; Stone, J C; Richardson, P M; Dunn, R J

    2001-01-01

    Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

  13. Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Skaar, Karin; Korza, Henryk J; Tarry, Michael; Sekyrova, Petra; Högbom, Martin

    2015-01-01

    Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies. PMID:26218426

  14. Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Karin Skaar

    Full Text Available Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid polymer technique for use in further biochemical or biophysical studies.

  15. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

    Science.gov (United States)

    Baghirova, Sabina; Hughes, Bryan G.; Hendzel, Michael J.; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C PMID:26740924

  16. Sub-cellular modeling of platelet transport in blood flow through microchannels with constriction.

    Science.gov (United States)

    Yazdani, Alireza; Karniadakis, George Em

    2016-05-11

    Platelet transport through arterial constrictions is one of the controlling processes influencing their adhesive functions and the formation of thrombi. We perform high-fidelity mesoscopic simulations of blood flow in microchannels with constriction, resembling arterial stenoses. The wall shear rates inside the constrictions reach levels as high as ≈8000 s(-1), similar to those encountered in moderate atherosclerotic plaques. Both red blood cells and platelets are resolved at sub-cellular resolution using the Dissipative Particle Dynamics (DPD) method. We perform a systematic study on the red blood cell and platelet transport by considering different levels of constriction, blood hematocrit and flow rates. We find that higher levels of constriction and wall shear rates lead to significantly enhanced margination of platelets, which may explain the experimental observations of enhanced post-stenosis platelet aggregation. We also observe similar margination effects for stiff particles of spherical shapes such as leukocytes. To our knowledge, such numerical simulations of dense blood through complex geometries have not been performed before, and our quantitative findings could shed new light on the associated physiological processes such as ATP release, plasma skimming, and thrombus formation. PMID:27087267

  17. Expression and subcellular localization of Ewing sarcoma (EWS) protein is affected by the methylation process.

    Science.gov (United States)

    Belyanskaya, Larisa L; Delattre, Olivier; Gehring, Heinz

    2003-08-15

    Ewing sarcoma (EWS) protein contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Recently, we had shown that EWS protein is not only localized in the nucleus and cytosol, but also on the surface of T cells and that its RBD is extensively asymmetrically dimethylated on arginine residues. Here we show that stimulation of T cells with phytohemagglutinin (PHA) caused a time-dependent 10-fold increase in expression of methylated EWS protein on the cell surface and a sixfold increase in the nuclei of peripheral blood mononuclear cells (PBMC). Mitogenic stimulation of malignant T cell lines, however, did not increase their inherently high expression of EWS protein. This expression seemed to correlate with methionine adenosyltransferase activity and S-adenosyl-L-methionine (AdoMet) utilization in PBMC and tumor cells and thus indicates dependence on the methylation process. Inhibition of methylation in normal and malignant cells with the methylation inhibitor adenosine dialdehyde (AdOx) resulted in a three to fivefold decreased expression of EWS protein not only in the nucleus but also on the cell surface. The inhibitory effect of AdOx was compensated and negligible in PBMC, but not in tumor cells if they were treated simultaneously with mitogenic PHA concentrations. The present findings indicate that expression of EWS protein in the various subcellular compartments is affected by the methylation process, in particular by the availability of intracellular AdoMet.

  18. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    Science.gov (United States)

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  19. Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance.

    Science.gov (United States)

    Di Maso, Vittorio; Avellini, Claudio; Crocè, Lory Saveria; Rosso, Natalia; Quadrifoglio, Franco; Cesaratto, Laura; Codarin, Erika; Bedogni, Giorgio; Beltrami, Carlo Alberto; Tell, Gianluca; Tiribelli, Claudio

    2007-01-01

    APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.

  20. Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

    Science.gov (United States)

    Liapis, Evangelos; Sandiford, Simone; Wang, Qiang; Gaidosh, Gabriel; Motti, Dario; Levay, Konstantin; Slepak, Vladlen Z

    2012-08-01

    The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

  1. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  2. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    Science.gov (United States)

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  3. Monte-Carlo study of energy deposition by heavy charged particles in sub-cellular volumes

    International Nuclear Information System (INIS)

    Detailed-history Monte-Carlo code is used to study the energy deposition from proton and alpha particle tracks at the sub-cellular level. Inelastic cross sections for both the vapour and liquid phases of water have been implemented into the code in order to explore the influence of non-linear density effects associated with the condensed-phase cellular environment. Results of energy deposition and its straggling for 0.5 to 5 MeV amu-1 protons and alpha particles traversing or passing near spherical volumes of 2-200 nm in diameter relevant to DNA- and chromosome-size targets are presented. It is shown that the explicit account of δ-ray transport reduces the dose by as much as 10-60%, whereas stochastic fluctuations lead to a relative uncertainty ranging from 20% to more than 100%. Protons and alpha particles of the same velocity exhibit a similar δ-ray effect, whereas the relative uncertainty of the alphas is almost half that of protons. The effect of the phase is noticeable (10-15%) mainly through differences on the transport of δ-rays, which in liquid water have higher penetration distances. It is expected that the implementation of such results into multi-scale biophysical models of radiation effects will lead to a more realistic predictions on the efficacy of new radiotherapeutic modalities that employ either external proton beam irradiation or internal alpha-emitting radionuclides. (authors)

  4. Subcellular distribution and chemical forms of cadmium in Impatiens walleriana in relation to its phytoextraction potential.

    Science.gov (United States)

    Lai, Hung-Yu

    2015-11-01

    Impatiens (Impatiens walleriana) has been shown to be a potential cadmium (Cd) hyperaccumulator, but its mechanisms in accumulation and detoxification have not been reported. Rooted cuttings of Impatiens were planted in artificially Cd-contaminated soils for 50 days with total target concentrations of 0, 10, 20, 40, 80, and 120 mg/kg. The subcellular distribution and chemical forms of Cd in the different organs were analyzed after the pot experiment. Compared with the control group, various Cd treatments affected the growth exhibitions of Impatiens, but most of them were not statistically significant. The Cd accumulation of different organs increased with an increase in the soil Cd concentrations for most of the treatments, and it was in the decreasing order of root>stem>leaf. In the roots of Impatiens, Cd was mainly compartmentalized in the soluble fraction (Fs), which has a high migration capacity and will further translocate to the shoot. The Cd was mainly compartmentalized in the cell wall fraction (Fcw) in the shoots as a mechanism of tolerance. Most of the Cd in the various organs of Impatiens was mainly in the forms of pectate and protein-integrated (FNaCl), whereas a minor portion was a water soluble fraction (FW). The experimental results show that the Cd in the Fs, FW, and FNaCl in the roots of Impatiens had a high mobility and will further translocate to the shoot. They could be used to estimate the Cd accumulated in the shoots of Impatiens.

  5. Subcellular clearance and accumulation of Huntington disease protein: A mini-review

    Directory of Open Access Journals (Sweden)

    Ting eZhao

    2016-04-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ tract in the N-terminal region of mutant huntingtin (mHtt. As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies of examining mHtt clearance and accumulation in different subcellular regions

  6. Subcellular object quantification with Squassh3C and SquasshAnalyst.

    Science.gov (United States)

    Rizk, Aurélien; Mansouri, Maysam; Ballmer-Hofer, Kurt; Berger, Philipp

    2015-11-01

    Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst. Squassh3C extends the functionality of Squassh to three fluorescence channels and live-cell movie analysis. SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. It provides segmentation image overview and data exploration, figure generation, object and image filtering, and a statistical significance test in an easy-to-use interface. The overall procedure combines the Squassh3C plug-in for the free biological image processing program ImageJ and a web application working in conjunction with the free statistical environment R, and it is compatible with Linux, MacOS X, or Microsoft Windows. Squassh3C and SquasshAnalyst are available for download at www.psi.ch/lbr/SquasshAnalystEN/SquasshAnalyst.zip. PMID:26554508

  7. Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans.

    Science.gov (United States)

    Lin, Ching-Wen; Wang, Po-Hsiang; Ismail, Wael; Tsai, Yu-Wen; El Nayal, Ashraf; Yang, Chia-Ying; Yang, Fu-Chun; Wang, Chia-Hsiang; Chiang, Yin-Ru

    2015-01-01

    Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a β-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

  8. Localization and subcellular distribution of N-copine in mouse brain.

    Science.gov (United States)

    Nakayama, T; Yaoi, T; Kuwajima, G

    1999-01-01

    N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+-dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+-dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of "double C2 domain-containing proteins," including synaptotagmin I, in presynaptic events. PMID:9886090

  9. The Impact of BK Channels on Cellular Excitability Depends on their Subcellular Location

    Science.gov (United States)

    Bock, Tobias; Stuart, Greg J.

    2016-01-01

    Large conductance calcium-activated potassium channels (or BK channels) fulfil a multitude of roles in the central nervous system. At the soma of many neuronal cell types they control the speed of action potential (AP) repolarization and therefore they can have an impact on neuronal excitability. Due to their presence in nerve terminals they also regulate transmitter release. BK channels have also been shown to be present in the dendrites of some neurons where they can regulate the magnitude and duration of dendritic spikes. Here, we investigate the impact of modulating the activation of BK channels at different locations on the cellular excitability of cortical layer 5 pyramidal neurons. We find that while somatic BK channels help to repolarize APs at the soma and mediate the fast after-hyperpolarization, dendritic BK channels are responsible for repolarization of dendritic calcium spikes and thereby regulate somatic AP burst firing. We found no evidence for a role of dendritic BK channels in the regulation of backpropagating AP amplitude or duration. These experiments highlight the diverse roles of BK channels in regulating neuronal excitability and indicate that their functional impact depends on their subcellular location.

  10. Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

    Science.gov (United States)

    Zheng, Wu; Blake, Catherine

    2015-10-01

    Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts. PMID:26220461

  11. Subcellular compartmentalization of docking protein-1 contributes to progression in colorectal cancer.

    Science.gov (United States)

    Friedrich, Teresa; Söhn, Michaela; Gutting, Tobias; Janssen, Klaus-Peter; Behrens, Hans-Michael; Röcken, Christoph; Ebert, Matthias P A; Burgermeister, Elke

    2016-06-01

    Full-length (FL) docking protein-1 (DOK1) is an adapter protein which inhibits growth factor and immune response pathways in normal tissues, but is frequently lost in human cancers. Small DOK1 variants remain in cells of solid tumors and leukemias, albeit, their functions are elusive. To assess the so far unknown role of DOK1 in colorectal cancer (CRC), we generated DOK1 mutants which mimic the domain structure and subcellular distribution of DOK1 protein variants in leukemia patients. We found that cytoplasmic DOK1 activated peroxisome-proliferator-activated-receptor-gamma (PPARγ) resulting in inhibition of the c-FOS promoter and cell proliferation, whereas nuclear DOK1 was inactive. PPARγ-agonist increased expression of endogenous DOK1 and interaction with PPARγ. Forward translation of this cell-based signaling model predicted compartmentalization of DOK1 in patients. In a large series of CRC patients, loss of DOK1 protein was associated with poor prognosis at early tumor stages (*p=0.001; n=1492). In tumors with cytoplasmic expression of DOK1, survival was improved, whereas nuclear localization of DOK1 correlated with poor outcome, indicating that compartmentalization of DOK1 is critical for CRC progression. Thus, DOK1 was identified as a prognostic factor for non-metastatic CRC, and, via its drugability by PPARγ-agonist, may constitute a potential target for future cancer treatments. PMID:27428427

  12. Effects of carbohydrate supplements on exercise-induced menstrual dysfunction and ovarian subcellular structural changes in rats

    Directory of Open Access Journals (Sweden)

    Can Zhao

    2014-09-01

    Conclusion: Female adult rats with 9-week continuous exercise can cause menstrual dysregulation as a model for EAMD. Post-EAMD intervention with glucose and oligosaccharide intake can normalize the menstrual cycle, restore the follicular subcellular structure, and reverse the exercise-induced reduction of ovary sex hormones. It suggests a positive feedback of hypothalamus–pituitary–ovary axis might be involved in the molecular mechanisms of energy intake in treating EAMD.

  13. Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products

    OpenAIRE

    Cummins, Timothy D.; Higdon, Ashlee N; Kramer, Philip A.; Chacko, Balu K; Riggs, Daniel W.; Salabei, Joshua K.; Dell’Italia, Louis J.; Zhang, Jianhua; Darley-Usmar, Victor M.; Hill, Bradford G.

    2012-01-01

    Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled...

  14. Huntingtin Subcellular Localisation Is Regulated by Kinase Signalling Activity in the StHdhQ111 Model of HD

    OpenAIRE

    Kathryn R Bowles; Brooks, Simon P.; Dunnett, Stephen B.; Lesley Jones

    2015-01-01

    Huntington’s disease is a neurodegenerative disorder characterised primarily by motor abnormalities, and is caused by an expanded polyglutamine repeat in the huntingtin protein. Huntingtin dynamically shuttles between subcellular compartments, and the mutant huntingtin protein is mislocalised to cell nuclei, where it may interfere with nuclear functions, such as transcription. However, the mechanism by which mislocalisation of mutant huntingtin occurs is currently unknown. An immortalised emb...

  15. MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria.

    Science.gov (United States)

    Magnus, Marcin; Pawlowski, Marcin; Bujnicki, Janusz M

    2012-12-01

    Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction. PMID:22705560

  16. PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

    Institute of Scientific and Technical Information of China (English)

    Evangelia I. Petsalaki; Pantelis G. Bagos; Zoi I. Litou; Stavros J. Hamodrakas

    2006-01-01

    The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function.We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast,thylakoid, mitochondrion, secretory pathway, and "other". When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:http://bioinformatics.biol.uoa.gr/PredSL/.

  17. Precision automation of cell type classification and sub-cellular fluorescence quantification from laser scanning confocal images

    Directory of Open Access Journals (Sweden)

    Hardy Craig Hall

    2016-02-01

    Full Text Available While novel whole-plant phenotyping technologies have been successfully implemented into functional genomics and breeding programs, the potential of automated phenotyping with cellular resolution is largely unexploited. Laser scanning confocal microscopy has the potential to close this gap by providing spatially highly resolved images containing anatomic as well as chemical information on a subcellular basis. However, in the absence of automated methods, the assessment of the spatial patterns and abundance of fluorescent markers with subcellular resolution is still largely qualitative and time-consuming. Recent advances in image acquisition and analysis, coupled with improvements in microprocessor performance, have brought such automated methods within reach, so that information from thousands of cells per image for hundreds of images may be derived in an experimentally convenient time-frame. Here, we present a MATLAB-based analytical pipeline to 1 segment radial plant organs into individual cells, 2 classify cells into cell type categories based upon random forest classification, 3 divide each cell into sub-regions, and 4 quantify fluorescence intensity to a subcellular degree of precision for a separate fluorescence channel. In this research advance, we demonstrate the precision of this analytical process for the relatively complex tissues of Arabidopsis hypocotyls at various stages of development. High speed and robustness make our approach suitable for phenotyping of large collections of stem-like material and other tissue types.

  18. Subcellular distribution of trace elements and liver histology of landlocked Arctic char (Salvelinus alpinus) sampled along a mercury contamination gradient.

    Science.gov (United States)

    Barst, Benjamin D; Rosabal, Maikel; Campbell, Peter G C; Muir, Derek G C; Wang, Xioawa; Köck, Günter; Drevnick, Paul E

    2016-05-01

    We sampled landlocked Arctic char (Salvelinus alpinus) from four lakes (Small, 9-Mile, North, Amituk) in the Canadian High Arctic that span a gradient of mercury contamination. Metals (Hg, Se, Tl, and Fe) were measured in char tissues to determine their relationships with health indices (relative condition factor and hepatosomatic index), stable nitrogen isotope ratios, and liver histology. A subcellular partitioning procedure was employed to determine how metals were distributed between potentially sensitive and detoxified compartments of Arctic char livers from a low- and high-mercury lake (Small Lake and Amituk Lake, respectively). Differences in health indices and metal concentrations among char populations were likely related to differences in feeding ecology. Concentrations of Hg, Se, and Tl were highest in the livers of Amituk char, whereas concentrations of Fe were highest in Small and 9-Mile char. At the subcellular level we found that although Amituk char had higher concentrations of Tl in whole liver than Small Lake char, they maintained a greater proportion of this metal in detoxified fractions, suggesting an attempt at detoxification. Mercury was found mainly in potentially sensitive fractions of both Small and Amituk Lake char, indicating that Arctic char are not effectively detoxifying this metal. Histological changes in char livers, mainly in the form of melano-macrophage aggregates and hepatic fibrosis, could be linked to the concentrations and subcellular distributions of essential or non-essential metals.

  19. Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase

    Directory of Open Access Journals (Sweden)

    Dugas Sandra L

    2003-07-01

    Full Text Available Abstract Background The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. Results Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the γ-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (γ-Proteobacteria possess similar plasmid-encoded arsC sequences. Conclusion The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.

  20. Archaeal Viruses Contribute to the Novel Viral Assemblage Inhabiting Oceanic, Basalt-Hosted Deep Subsurface Crustal Fluids

    Science.gov (United States)

    Nigro, O. D.; Rappe, M. S.; Jungbluth, S.; Lin, H. T.; Steward, G.

    2015-12-01

    Fluids contained in the basalt-hosted deep subsurface of the world's oceans represent one of the most inaccessible and understudied biospheres on earth. Recent improvements in sampling infrastructure have allowed us to collect large volumes of crustal fluids (~104 L) from Circulation Obviation Retrofit Kits (CORKs) placed in boreholes located on the eastern flank of the Juan de Fuca Ridge (JdFR). We detected viruses within these fluids by TEM and epifluorescence microscopy in samples collected from 2010 to 2014. Viral abundance, determined by epifluorescence counts, indicated that concentrations of viruses in subsurface basement fluids (~105 ml-1) are lower than the overlying seawater, but are higher in abundance than microbial cells in the same samples. Analysis of TEM images revealed distinct viral morphologies (rod and spindle-shaped) that resemble the morphologies of viral families infecting archaea. There are very few, if any, direct observations of these viral morphologies in marine samples, although they have been observed in enrichment cultures and their signature genes detected in metagenomic studies from hydrothermal vents and marine sediments. Analysis of metagenomes from the JdFR crustal fluids revealed sequences with homology to archaeal viruses from the rudiviridae, bicaudaviridae and fuselloviridae. Prokaryotic communities in fluids percolating through the basaltic basement rock of the JdFR flank are distinct from those inhabiting the overlying sediments and seawater. Similarly, our data support the idea that the viral assemblage in these fluids is distinct from viral assemblages in other marine and terrestrial aquatic environments. Our data also suggest that viruses contribute to the mortality of deep subsurface prokaryotes through cell lysis, and viruses may alter the genetic potential of their hosts through the processes of lysogenic conversion and horizontal gene transfer.

  1. Bacterial CS2 hydrolases from Acidithiobacillus thiooxidans strains are homologous to the archaeal catenane CS2 hydrolase.

    Science.gov (United States)

    Smeulders, Marjan J; Pol, Arjan; Venselaar, Hanka; Barends, Thomas R M; Hermans, John; Jetten, Mike S M; Op den Camp, Huub J M

    2013-09-01

    Carbon disulfide (CS(2)) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS(2) is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO(2)) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS(2)-polluted airstreams. We report on the mechanism of bacterial CS(2) conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS(2) hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS(2) hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of β-carbonic anhydrase (β-CA) homologues that may comprise a subclass of CS(2) hydrolases within the β-CA family. Unlike CAs, the CS(2) hydrolases did not hydrate CO(2) but converted CS(2) and COS with H(2)O to H(2)S and CO(2). The CS(2) hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS(2) hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS(2) hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS(2) hydrolases based on the structure of Acidianus strain A1-3 CS(2) hydrolase suggest that the A. thiooxidans strain G8 CS(2) hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.

  2. Archaeal Ammonia Oxidizers and Total Production of N2O and CH4 in Arctic Polar Desert Soils

    Science.gov (United States)

    Brummell, Martin; Robert, Stan; Bodrossy, Levente; Abell, Guy; Siciliano, Steven

    2014-05-01

    Ammonia-oxidizing Archaea are abundant in Arctic desert soils and appear to be responsible for the majority of ammonia oxidation activity in these cold and dry ecosystems. We used DNA microarrays to characterize the microbial community consisting of ammonia-oxidizing Archaea and methane-oxidizing Bacteria in three polar deserts from Ellesmere Island, Canada. Patterns of net greenhouse gas production, including production and consumption of CO2, CH4, and N2O were compared with community relative richness and abundance in a structural equation model that tested causal hypotheses relating edaphic factors to the biological community and net gas production. We extracted and amplified DNA sequences from soils collected at three polar deserts on Ellesmere Island in the Canadian high Arctic, and characterized the community structure using DNA microarrays. The functional genes Archaeal AmoA and pMMO were used to compare patterns of biological community structure to the observed patterns of net greenhouse gas production from those soils, as measured in situ. Edaphic factors including water content, bulk density, pH, and nutrient levels such as nitrate, ammonia, and extractable organic carbon were also measured for each soil sample, resulting in a highly multivariate dataset. Both concentration and net production of the three greenhouse gases were correlated, suggesting underlying causal factors. Edaphic factors such as soil moisture and pH had important, direct effects on the community composition of both functional groups of microorganisms, and pH further had a direct effect on N2O production. The structural relationship between the examined microbial communities and net production of both N2O and CH4 was strong and consistent between varying model structures and matrices, providing high confidence that this model relationship accurately reflects processes occurring in Arctic desert soils.

  3. Archaeal and bacterial diversity in an arsenic-rich shallow-sea hydrothermal system undergoing phase separation

    Directory of Open Access Journals (Sweden)

    Roy Edward Price

    2013-07-01

    Full Text Available Phase separation is a ubiquitous process in seafloor hydrothermal vents, creating a large range of salinities. Toxic elements (e.g., arsenic partition into the vapor phase, and thus can be enriched in both high and low salinity fluids. However, investigations of microbial diversity at sites associated with phase separation are rare. We evaluated prokaryotic diversity in arsenic-rich shallow-sea vents off Milos Island (Greece by comparative analysis of 16S rRNA clone sequences from two vent sites with similar pH and temperature but marked differences in salinity. Clone sequences were also obtained for aioA-like functional genes (AFGs. Bacteria in the surface sediments (0 to 1.5 cm at the high salinity site consisted of mainly Epsilonproteobacteria (Arcobacter sp., which transitioned to almost exclusively Firmicutes (Bacillus sp. at ~10 cm depth. However, the low salinity site consisted of Bacteroidetes (Flavobacteria in the surface and Epsilonproteobacteria (Arcobacter sp. at ~10 cm depth. Archaea in the high salinity surface sediments were dominated by the orders Archaeoglobales and Thermococcales, transitioning to Thermoproteales and Desulfurococcales (Staphylothermus sp. in the deeper sediments. In contrast, the low salinity site was dominated by Thermoplasmatales in the surface and Thermoproteales at depth. Similarities in gas and redox chemistry suggest that salinity and/or arsenic concentrations may select for microbial communities that can tolerate these parameters. Many of the archaeal 16S rRNA sequences contained inserts, possibly introns, including members of the Euryarchaeota. Clones containing AFGs affiliated with either Alpha- or Betaproteobacteria, although most were only distantly related to published representatives. Most clones (89% originated from the deeper layer of the low salinity, highest arsenic site. This is the only sample with overlap in 16S rRNA data, suggesting arsenotrophy as an important metabolism in similar

  4. Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

    OpenAIRE

    Pufeng Du; Lusheng Wang

    2014-01-01

    One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as w...

  5. The subcellular distribution of 239Pu, 241Am and 59Fe in the liver of rat and Chinese hamster as dependent on time

    International Nuclear Information System (INIS)

    The subcellular distribution of monomeric 239Pu, 241Am and iron in rat and Chinese hamster liver has been investigated by sucrose-, metrizamide- and Percoll-density gradients. In rat liver, the transuranium elements become and remain bound to typical lysosomes primary storage organelle in Chinese hamster liver. However, their apparent density in sucrose decreases with time, which possible indicates transition into telolysomes. The transuranium nuclides show a subcellular distribution which is quite different from that of iron. (orig.)

  6. The sub-cellular fate of mercury in the liver of wild mullets (Liza aurata) – Contribution to the understanding of metal-induced cellular toxicity

    International Nuclear Information System (INIS)

    Highlights: • This study is one of the first reports of Hg sub-cellular partitioning in wild fish. • Hg partition in Liza aurata liver differed between reference and contaminated sites. • Levels of Hg in sub-cellular fractions were in line with environmental contamination. • Lysosomes plus microsomes was the main fraction bounding Hg at the contaminated site. • Sub-cellular partitioning of Hg revealed to be a promising indicator of cellular toxicity. - Abstract: Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas – contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys + Mic). At LAR, Lys + Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions

  7. Spatial Variations in Archaeal Lipids of Surface Water and Core-Top Sediments in the South China Sea and Their Implications for Paleoclimate Studies▿†

    Science.gov (United States)

    Wei, Yuli; Wang, Jinxiang; Liu, Jie; Dong, Liang; Li, Li; Wang, Hui; Wang, Peng; Zhao, Meixun; Zhang, Chuanlun L.

    2011-01-01

    The South China Sea (SCS) is the largest marginal sea of the western Pacific Ocean, yet little is known about archaeal distributions and TEX86-based temperatures in this unique oceanic setting. Here we report findings of abundances in both core lipids (CL) and intact polar lipids (IPL) of Archaea from surface water (CL only) and core-top sediments from different regions of the SCS. TEX86-derived temperatures were also calculated for these samples. The surface water had extremely low abundances of CL (average of 0.05 ± 0.13 ng/liter; n = 75), with higher values present in regions where upwelling is known to occur. The core-top sediments had CL values of 0.1 to 0.9 μg/g, which are on the low end of CL concentrations reported for other marine sediments and may reflect the oligotrophic nature of the open SCS. The IPL of Archaea accounted for 6 to 36.4% of total lipids (CL plus IPL), indicating that the majority of archaeal lipids in core-top sediments were derived from nonliving cells. The TEX86-based temperatures of surface water were overall lower than satellite-based sea surface temperatures or CTD-measured in situ temperatures. The core-top sediment samples, however, had TEX86 temperatures very close to the mean annual sea surface temperatures, except for samples with water depths of less than 100 m. Our results demonstrated low and heterogeneous distributions of archaeal lipids in surface water and core-top sediments of the SCS, which may reflect local or regional differences in productivity of Archaea. While TEX86-based temperatures for core-top marine sediments at deep water depths (>100 m) generally reflected mean annual sea surface temperatures, TEX86 temperatures in surface water varied basin wide and underestimated sea surface temperatures in most locations for the season when surface water samples were collected. PMID:21890672

  8. Novel PCR primers for the archaeal phylum Thaumarchaeota designed based on the comparative analysis of 16S rRNA gene sequences.

    Directory of Open Access Journals (Sweden)

    Jin-Kyung Hong

    Full Text Available Based on comparative phylogenetic analysis of 16S rRNA gene sequences deposited in an RDP database, we constructed a local database of thaumarchaeotal 16S rRNA gene sequences and developed a novel PCR primer specific for the archaeal phylum Thaumarchaeota. Among 9,727 quality-filtered (chimeral-checked, size >1.2 kb archaeal sequences downloaded from the RDP database, 1,549 thaumarchaeotal sequences were identified and included in our local database. In our study, Thaumarchaeota included archaeal groups MG-I, SAGMCG-I, SCG, FSCG, RC, and HWCG-III, forming a monophyletic group in the phylogenetic tree. Cluster analysis revealed 114 phylotypes for Thaumarchaeota. The majority of the phylotypes (66.7% belonged to the MG-I and SCG, which together contained most (93.9% of the thaumarchaeotal sequences in our local database. A phylum-directed primer was designed from a consensus sequence of the phylotype sequences, and the primer's specificity was evaluated for coverage and tolerance both in silico and empirically. The phylum-directed primer, designated THAUM-494, showed >90% coverage for Thaumarchaeota and 95% of the amplified sequences belonged to Thaumarchaeota. The most frequently sampled thaumarchaeotal subgroups in our samples were SCG, MG-I, and SAGMCG-I. To our knowledge, THAUM-494 is the first phylum-level primer for Thaumarchaeota. Furthermore, the high coverage and low tolerance of THAUM-494 will make it a potentially valuable tool in understanding the phylogenetic diversity and ecological niche of Thaumarchaeota.

  9. Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers.

    Science.gov (United States)

    Niu, Yuhong; Meng, Qingxiang; Li, Shengli; Ren, Liping; Zhou, Bo; Schonewille, Thomas; Zhou, Zhenming

    2016-01-01

    This study investigated the effects of ensiled mulberry leaves (EML) and sun-dried mulberry fruit pomace (SMFP) on the ruminal bacterial and archaeal community composition of finishing steers. Corn grain- and cotton meal-based concentrate was partially replaced with EML or SMFP. The diets had similar crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy. Following the feeding trial, the steers were slaughtered and ruminal liquid samples were collected to study the ruminal microbiome. Extraction of DNA, amplification of the V4 region of the 16S rRNA gene, and Illumina MiSeq pyrosequencing were performed for each sample. Following sequence de-noising, chimera checking, and quality trimming, an average of 209,610 sequences were generated per sample. Quantitative real-time PCR was performed to examine the selected bacterial species in the rumen. Our results showed that the predominant phyla were Bacteroidetes (43.90%), Firmicutes (39.06%), Proteobacteria (4.31%), and Tenericutes (2.04%), and the predominant genera included Prevotella (13.82%), Ruminococcus (2.51%), Butyrivibrio (2.38%), and Succiniclasticum (2.26%). Compared to the control group, EML and SMFP groups had a higher abundance of total bacteria (p composition was similar among the three groups. At the phylum level, there were no significant differences in Firmicutes (p = 0.7932), Bacteroidetes (p = 0.2330), Tenericutes (p = 0.2811), or Proteobacteria (p = 0.0680) levels among the three groups; however, Fibrobacteres decreased in EML (p = 0.0431). At the genus level, there were no differences in Prevotella (p = 0.4280), Ruminococcus (p = 0.2639), Butyrivibrio (p = 0.4433), or Succiniclasticum (p = 0.0431) levels among the groups. Additionally, the dietary treatments had no significant effects on the archaeal community composition in the rumen. Therefore, EML and SMFP supplementation had no significant effects on the ruminal bacterial or archaeal community composition of finishing steers.

  10. Temperature increases from 55 to 75 C in a two-phase biogas reactor result in fundamental alterations within the bacterial and archaeal community structure

    Energy Technology Data Exchange (ETDEWEB)

    Rademacher, Antje [Leibniz-Institut fuer Agrartechnik Potsdam-Bornim e.V. (ATB), Potsdam (Germany). Abt. Bioverfahrenstechnik; Technische Univ. Berlin (Germany). Inst. fuer Technischen Umweltschutz; Nolte, Christine; Schoenberg, Mandy; Klocke, Michael [Leibniz-Institut fuer Agrartechnik Potsdam-Bornim e.V. (ATB), Potsdam (Germany). Abt. Bioverfahrenstechnik

    2012-10-15

    Agricultural biogas plants were operated in most cases below their optimal performance. An increase in the fermentation temperature and a spatial separation of hydrolysis/acetogenesis and methanogenesis are known strategies in improving and stabilizing biogas production. In this study, the dynamic variability of the bacterial and archaeal community was monitored within a two-phase leach bed biogas reactor supplied with rye silage and straw during a stepwise temperature increase from 55 to 75 C within the leach bed reactor (LBR), using TRFLP analyses. To identify the terminal restriction fragments that were obtained, bacterial and archaeal 16S rRNA gene libraries were constructed. Above 65 C, the bacterial community structure changed from being Clostridiales-dominated toward being dominated by members of the Bacteroidales, Clostridiales, and Thermotogales orders. Simultaneously, several changes occurred, including a decrease in the total cell count, degradation rate, and biogas yield along with alterations in the intermediate production. A bioaugmentation with compost at 70 C led to slight improvements in the reactor performance; these did not persist at 75 C. However, the archaeal community within the downstream anaerobic filter reactor (AF), operated constantly at 55 C, altered by the temperature increase in the LBR. At an LBR temperature of 55 C, members of the Methanobacteriales order were prevalent in the AF, whereas at higher LBR temperatures Methanosarcinales prevailed. Altogether, the best performance of this two-phase reactor was achieved at an LBR temperature of below 65 C, which indicates that this temperature range has a favorable effect on the microbial community responsible for the production of biogas. (orig.)

  11. Archaeal and Bacterial Diversity and Enzymatic Activities Associated With Particulate Matter in the Laptev Sea, a River-Impacted Arctic Shelf Environment

    Science.gov (United States)

    Evans, C. T.; Deming, J. W.

    2006-12-01

    Arctic Ocean shelves are influenced by riverine input of terrestrial, relatively refractory particulate organic matter (POM) as well as fresh material from marine phytoplankton blooms. The fate of organic particles and aggregates depends in large part on their associated microbes and the effectiveness of hydrolytic enzymes. The Laptev Sea provides an ideal setting to test for connections between Archaeal and Bacterial communities, the quality of the POM they colonize, and the activities of extracellular enzymes. Aboard the Russian icebreaker Kapitan Dranitsyn during the NABOS 2005 cruise to the Laptev Sea, we sampled various size fractions of particulate matter, from 0.2 to 70 μm. Patterns of Archaeal and Bacterial diversity were analyzed using terminal restriction fragment length polymorphism (T-RFLP). Extracellular enzymatic activities were evaluated using fluorescent substrate analogs. Thus far, we have observed a statistically significant difference between particle-associated and free-living Bacteria, many of which appear (by clone library) to be gamma-proteobacteria or CFB. Bacterial community richness associated with the largest particle fractions, where protease and glucosidase activities were the highest, was best explained by indicators of primary productivity (chlorophyll a and phaeopigments), while richness associated with smaller size fractions was best explained by general particle indicators (and depth and salinity). In contrast, particle-associated Archaea were not significantly different from their free-living counterparts. Archaeal clone library results indicate a predominance of Marine Group 1 Crenarchaea, the group containing a recently isolated nitrifying Archaeon. Given all these results, we hypothesize that in the Laptev Sea cold-active Bacteria are the primary agents in the enzymatic degradation of POM, whether terrestrial or marine, while Archaea play other roles in the elemental cycles of Arctic waters, perhaps especially in the nitrogen

  12. Pyrosequencing reveals the influence of elevated atmospheric CO2 on the composition of archaeal communities in the rhizosphere of C3 and C4 crops

    Science.gov (United States)

    Nelson, D. M.; Cann, I. K.; Mackie, R. I.

    2008-12-01

    The projected increase in atmospheric CO2 concentrations throughout the 21st century is likely to increase aboveground and belowground plant productivity and cause changes in the quantity and quality of plant root exudates, although plants using C4 photosynthesis are likely to be only affected during times of drought (Leakey et al., 2006, Plant Physiology, 140, 779). Evidence is emerging from molecular tools that these changes may influence the abundance and composition of soil microbial communities that regulate key soil processes, such as nitrogen cycling (Lesaulnier et al., 2008, Environmental Microbiology, 10, 926). However, most molecular tools are not well-suited for comparing multiple samples at great sequencing depth, which is critical when considering soil microbial communities of high diversity. To overcome these limitations we used pyrosequencing and quantitative PCR (qPCR) of two genes (the V3 region of 16S rDNA and the amoA gene) to examine intra- and inter-treatment variability in the abundance and composition of microbial communities in the rhizosphere of soybean (C3) and maize (C4) grown in field conditions under ambient (~380 ppm) and elevated (~550 ppm) CO2 using FACE (free-air concentration enrichment) technology during the 2006 growing season in central Illinois. We specifically focused on archaeal communities because of their key role in nitrification (Leininger et al., 2006, Nature, 442, 806). The majority (>97%) of recovered sequences were from members of the phylum Crenarchaeota. Principle component analysis of sequence results from the V3 and amoA genes indicated significant (psoybean, but not maize. qPCR suggested no significant difference in the abundance of archaea between treatments for soybean and maize. The lack of response of archaeal community composition beneath maize to elevated CO2 is consistent with relatively high soil moisture availability throughout the summer of 2006, whereas the significant influence of elevated CO2 on

  13. Algal and archaeal polyisoprenoids in a recent marine sediment: Molecular isotopic evidence for anaerobic oxidation of methane RID C-7675-2009

    DEFF Research Database (Denmark)

    Bian, LQ; Hinrichs, KU; Xie, TM;

    2001-01-01

    Analyses of C-13 contents of individual organic molecules in a marine sediment show that crocetane, 2,6,11,15-tetramethylhexadecane, an isomer of phytane, is produced by microorganisms that use methane as their main source of carbon. The sediments lie at a water depth of 68 m in the Kattegat......-consuming member of the microbial consortium responsible for the anaerobic oxidation of methane [Hoehler et al., 1994], in which, as first demonstrated quantitatively in these sediments [Iversen and Jørgensen, 1985], electrons are transferred from methane to sulfate. The presence of archaeal biomass throughout...

  14. Combined phase and X-Ray fluorescence imaging at the sub-cellular level

    International Nuclear Information System (INIS)

    This work presents some recent developments in the field of hard X-ray imaging applied to biomedical research. As the discipline is evolving quickly, new questions appear and the list of needs becomes bigger. Some of them are dealt with in this manuscript. It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimental setup to investigate diverse aspects of cellular research. Together with its high spatial resolution, high flux and high energy range the experimental setup provides bigger field of view, is less sensitive to radiation damages (while taking phase contrast images) and suits well chemical analysis with emphasis on endogenous metals (Zn, Fe, Mn) but also with a possibility for exogenous one's like these found in nanoparticles (Au, Pt, Ag) study. Two synchrotron-based imaging techniques, fluorescence and phase contrast imaging were used in this research project. They were correlated with each other on a number of biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA, red blood cells). The explorations made in the chapter 5 allowed preparation of more established and detailed analysis, described in the next chapter where both techniques, X-ray fluorescence and phase contrast imaging, were exploited in order to access absolute metal projected mass fraction in a whole cell. The final image presents for the first time true quantitative information at the sub-cellular level, not biased by the cell thickness. Thus for the first time a fluorescence map serves as a complete quantitative image of a cell without any risk of misinterpretation. Once both maps are divided by each other pixel by pixel (fluorescence map divided by the phase map) they present a complete and final result of the metal (Zn in this work) projected mass fraction in ppm of dry weight. For the purpose of this calculation the analysis was extended to calibration (non-biological) samples. Polystyrene spheres of a known diameter and known

  15. Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions.

    Science.gov (United States)

    Valente, Valeria; Maia, Rafaela Martins; Vianna, Murilo Carlos Bizam; Paçó-Larson, Maria Luisa

    2010-11-01

    Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family. PMID:20977671

  16. Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

    Science.gov (United States)

    Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

    2016-08-01

    Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed. PMID:26820914

  17. Subcellular distribution of uranium in the roots of Spirodela punctata and surface interactions

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Xiaoqin, E-mail: xiaoqin_nie@163.com [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Key Laboratory of Radiation Physics and Technology (Sichuan University), Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu 610064 (China); Dong, Faqin, E-mail: fqdong2004@163.com [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Liu, Ning [Key Laboratory of Radiation Physics and Technology (Sichuan University), Ministry of Education, Institute of Nuclear Science and Technology, Sichuan University, Chengdu 610064 (China); Liu, Mingxue [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China); Zhang, Dong; Kang, Wu [Institute of Nuclear Physics and Chemistry,China Academy of Engineering Physics, Mianyang 621900 (China); Sun, Shiyong; Zhang, Wei; Yang, Jie [Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Mianyang 621010 (China)

    2015-08-30

    Graphical abstract: - Highlights: • The proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. • The particles including 35% Fe (wt%) released from the cells after 100 mg/L U treatment 48 h. • Most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal. • FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed. - Abstract: The subcellular distribution of uranium in roots of Spirodela punctata (duckweed) and the process of surface interaction were studied upon exposure to U (0, 5–200 mg/L) at pH 5. The concentration of uranium in each subcelluar fraction increased significantly with increasing solution U level, after 200 mg/L uranium solution treatment 120 h, the proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. OM SEM and EDS showed after 5–200 mg/L U treatment 4–24 h, some intracellular fluid released from the root cells, after 100 mg/L U treatment 48 h, the particles including 35% Fe (wt%) and other organic matters such as EPS released from the cells, most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal, similar crystal has been found in the cell wall and organelle fractions after 50 mg/L U treatment 120 h. FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed.

  18. Arabinogalactan glycosyltransferases target to a unique subcellular compartment that may function in unconventional secretion in plants.

    Science.gov (United States)

    Poulsen, Christian Peter; Dilokpimol, Adiphol; Mouille, Grégory; Burow, Meike; Geshi, Naomi

    2014-11-01

    We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A [Arabidopsis thaliana galactosyltransferase from family 31 (At1g32930)] was found in the small compartments, of which, 45 and 40% of AtGALT29A [Arabidopsis thaliana galactosyltransferase from family 29 (At1g08280)] and AtGlcAT14A [Arabidopsis thaliana glucuronosyltransferase from family 14 (At5g39990)] colocalized with AtGALT31A, respectively; in contrast, N-glycosylation enzymes rarely colocalized (3-18%), implicating a role of the small compartments in a part of arabinogalactan (O-glycan) biosynthesis rather than N-glycan processing. The dual localization of AtGALT31A was also observed for fluorescently tagged AtGALT31A stably expressed in an Arabidopsis atgalt31a mutant background. Further, site-directed mutagenesis of a phosphorylation site of AtGALT29A (Y144) increased the frequency of the protein being targeted to the AtGALT31A-localized small compartments, suggesting a role of Y144 in subcellular targeting. The AtGALT31A localized to the small compartments were colocalized with neither SYP61 (syntaxin of plants 61), a marker for trans-Golgi network (TGN), nor FM4-64-stained endosomes. However, 41% colocalized with EXO70E2 (Arabidopsis thaliana exocyst protein Exo70 homolog 2), a marker for exocyst-positive organelles, and least affected by Brefeldin A and Wortmannin. Taken together, AtGALT31A localized to small compartments that are distinct from the Golgi apparatus, the SYP61-localized TGN, FM4-64-stained endosomes and Wortmannin-vacuolated prevacuolar compartments, but may be part of an unconventional protein secretory pathway represented by EXO70E2 in plants. PMID:25074762

  19. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  20. [Certain properties of "biosynthetic" L-threonine dehydratase from subcellular structures of brewers' yeast Saccharomyces carlsbergensis].

    Science.gov (United States)

    Kovaleva, S V; Korozhko, A I; Beliaeva, N F; Kagan, Z S

    1981-01-01

    The paper is concerned with kinetic properties of the "biosynthetic" L-threonine dehydratase (EC 4.2.1.16) solubilized from subcellular structures of brewers' yeast Saccharomyces carlsbergensis in the absence and presence of the allosteric inhibitor, L-isoleucine, at three pH-values (pH 6.5, 7.8 and 9.5). The curve of the initial reaction rate versus initial substrate concentration in the absence of L-isoleucine at pH 6.5 was of hyperbolic character (Km = 5.5.10(-2) M), and at pH 7.8 and 9.5 the kinetic curve had a weakly sigmoidal pattern with a sharp going into the saturation plateaux; the values of [S] 0.5 are 1.10(-2) and 8.7.10(-3) M, respectively. An addition of L-isoleucine to the reaction mixture led to the appearance (at pH 6.5) or to an increase (at pH 7.8 and 9.5) of the sigmoidality of these kinetic curves and to a decrease in values of the maximum reaction rate V. The enzyme sensibility to the inhibitory effect of L-isoleucine decreased with an increase in pH values. Low L-isoleucine concentrations at low substrate concentrations activated the enzyme. The pH optimum for L-threonine dehydratase under study was 9.5-10.0. The enzyme molecular weight is about 300 000.