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Sample records for archaeal orfeomes subcellular

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

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    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. 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

  4. 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

  5. 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...

  6. 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

  7. 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

  8. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

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    Albrecht von Brunn

    Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

  9. Archaeal virus-host interactions

    OpenAIRE

    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 high morphological and genetic diversity. Some archaeal viruses, such as Sulfolobus islandicus rod-shaped virus 2 (SIRV2), have quite remarkable infection cycles. As described in Chapter 1, infection ...

  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 with an...... 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 and the...

  11. 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

  12. Archaeal Enzymes and Applications in Industrial Biocatalysts

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    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.

  13. 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.

  14. 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.

  15. 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

  16. 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...

  17. Biosynthesis of archaeal membrane ether lipids.

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    Jain, Samta; Caforio, Antonella; Driessen, Arnold J M

    2014-01-01

    A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria. PMID:25505460

  18. 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...

  19. 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 for...... CRISPR loci and the evidence for intergenomic exchange of CRISPR systems....

  20. Structure and lability of archaeal dehydroquinase

    International Nuclear Information System (INIS)

    The structure and thermal melting data for dehydroquinase from A. fulgidus are reported. The protein melts in vitro well below the organism’s growth temperature. Multiple sequence alignments of type I 3-dehydroquinate dehydratases (DQs; EC 4.2.1.10) show that archaeal DQs have shorter helical regions than bacterial orthologs of known structure. To investigate this feature and its relation to thermostability, the structure of the Archaeoglobus fulgidus (Af) DQ dimer was determined at 2.33 Å resolution and its denaturation temperature was measured in vitro by circular dichroism (CD) and differential scanning calorimetry (DSC). This structure, a P212121 crystal form with two 45 kDa dimers in the asymmetric unit, is the first structural representative of an archaeal DQ. Denaturation occurs at 343 ± 3 K at both low and high ionic strength and at 349 K in the presence of the substrate analog tartrate. Since the growth optimum of the organism is 356 K, this implies that the protein maintains its folded state through the participation of additional factors in vivo. The (βα)8 fold is compared with those of two previously determined type I DQ structures, both bacterial (Salmonella and Staphylococcus), which had sequence identities of ∼30% with AfDQ. Although the overall folds are the same, there are many differences in secondary structure and ionic features; the archaeal protein has over twice as many salt links per residue. The archaeal DQ is smaller than its bacterial counterparts and lower in regular secondary structure, with its eight helices being an average of one turn shorter. In particular, two of the eight normally helical regions (the exterior of the barrel) are mostly nonhelical in AfDQ, each having only a single turn of 310-helix flanked by β-strand and coil. These ‘protohelices’ are unique among evolutionarily close members of the (βα)8-fold superfamily. Structural features that may contribute to stability, in particular ionic factors, are

  1. Archaeal transformation of metals in the environment.

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    Bini, Elisabetta

    2010-07-01

    We are becoming increasingly aware of the role played by archaea in the biogeochemical cycling of the elements. Metabolism of metals is linked to fundamental metabolic functions, including nitrogen fixation, energy production, and cellular processes based on oxidoreductions. Comparative genomic analyses have shown that genes for metabolism, resistance, and detoxification of metals are widespread throughout the archaeal domain. Archaea share with other organisms strategies allowing them to utilize essential metals and maintain metal ions within a physiological range, although comparative proteomics show, in a few cases, preferences for specific genetic traits related to metals. A more in-depth understanding of the physiology of acidophilic archaea might lead to the development of new strategies for the bioremediation of metal-polluted sites and other applications, such as biomining. PMID:20455933

  2. Hyperthermophilic Archaeal Viruses as Novel Nanoplatforms

    DEFF Research Database (Denmark)

    Uldahl, Kristine Buch

    applications, Chapter I presents an in depth investigation of the hyperthermophilic archaeal virus SMV. Decisive steps in the viral life-cycle are studied with focus on the early stages of infection. TEM observations suggest that SMV1 virions enter into host cells via a fusion entry mechanism, involving three...... increase therapeutic benefit and minimize adverse effects. Virus-based nanoplatforms take advantage of the natural circulatory and targeting properties of viruses, to design therapeutics that specifically target tissues of interest in vivo. Plant-based viruses and bacteriophages are typically considered...... distinct stages; 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...

  3. Subcellular localization of pituitary enzymes

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    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.

  4. 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.

  5. 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...

  6. The archaeal Sec-dependent protein translocation pathway.

    OpenAIRE

    Bolhuis, Albert

    2004-01-01

    Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems. One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life. Very little is known about this pathway in the third domain of life, archaea. The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts. Interest...

  7. 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

  8. 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 thr...

  9. A Survey of Protein Structures from Archaeal Viruses

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    Nikki Dellas

    2013-01-01

    Full Text Available Viruses that infect the third domain of life, Archaea, are a newly emerging field of interest. To date, all characterized archaeal viruses infect archaea that thrive in extreme conditions, such as halophilic, hyperthermophilic, and methanogenic environments. Viruses in general, especially those replicating in extreme environments, contain highly mosaic genomes with open reading frames (ORFs whose sequences are often dissimilar to all other known ORFs. It has been estimated that approximately 85% of virally encoded ORFs do not match known sequences in the nucleic acid databases, and this percentage is even higher for archaeal viruses (typically 90%–100%. This statistic suggests that either virus genomes represent a larger segment of sequence space and/or that viruses encode genes of novel fold and/or function. Because the overall three-dimensional fold of a protein evolves more slowly than its sequence, efforts have been geared toward structural characterization of proteins encoded by archaeal viruses in order to gain insight into their potential functions. In this short review, we provide multiple examples where structural characterization of archaeal viral proteins has indeed provided significant functional and evolutionary insight.

  10. Neuronal Computations Made Visible with Subcellular Resolution.

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    Kaschula, Richard; Salecker, Iris

    2016-06-30

    Sensory information is gradually processed within dedicated neural circuits to generate specific behaviors. In this issue, Yang et al. push technology boundaries to measure both voltage and calcium signals from subcellular compartments of genetically defined interconnected neurons and shed light on local neural computations critical for motion detection. PMID:27368098

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    JosephSteffens

    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

  13. 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...... 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....

  14. Bacterial and archaeal communities in Lake Nyos (Cameroon, Central Africa)

    OpenAIRE

    Tiodjio, Rosine E.; Sakatoku, Akihiro; Nakamura, Akihiro; Tanaka, Daisuke; Fantong, Wilson Y.; Tchakam, Kamtchueng B.; Tanyileke, Gregory; Ohba, Takeshi; Hell, Victor J.; Kusakabe, Minoru; Nakamura, Shogo; Ueda, Akira

    2014-01-01

    The aim of this study was to assess the microbial diversity associated with Lake Nyos, a lake with an unusual chemistry in Cameroon. Water samples were collected during the dry season on March 2013. Bacterial and archaeal communities were profiled using Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) approach of the 16S rRNA gene. The results indicate a stratification of both communities along the water column. Altogether, the physico-chemical data and microbial s...

  15. 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

  16. TBP domain symmetry in basal and activated archaeal transcription.

    Science.gov (United States)

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

    2009-01-01

    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 the Methanococcales and Thermoplasmatales, including complete conservation of their N- and C-terminal stirrups; along with helix H'1, the C-terminal stirrup of TBP forms the main interface with TFB/TFIIB. Here, we show that, in stark contrast to its eukaryotic counterparts, multiple substitutions in the C-terminal stirrup of Methanocaldococcus jannaschii (Mja) TBP do not completely abrogate basal transcription. Using DNA affinity cleavage, we show that, by assembling TFB through its conserved N-terminal stirrup, Mja TBP is in effect ambidextrous with regard to basal transcription. In contrast, substitutions in either its N- or the C-terminal stirrup abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2. PMID:19007415

  17. 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.

  18. 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.

  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. Subcellular distribution of curium in beagle liver

    International Nuclear Information System (INIS)

    The subcellular distribution of curium (243244Cm) was studied in canine liver from 2 hr to 47 days after injection of 3 μCi243244Cm/kg of body weight. The pattern of distribution for Cm was similar to other trivalent actinide elements studied previously (Am, Cf). Initially (2 hr), most of the nuclide was found in the cytosol and at least 90 percent was protein bound. About 70 percent of the Cm was bound to ferritin, approximately 5 percent was associated with a protein of MW approximately 200,000, and approximately 25 percent was found in the low-molecular-weight region (approximately 5000). The decrease in the Cm content of cytosol, nuclei, and microsomes coincided with an increase in the amount associated with mitochondria and lysosomes. The concentration of the Cm in the mitochondrial fraction was higher than it was in the lysosomal fraction at each time studied. In the mitochondrial fraction approximately 30 percent of the Cm was bound to membranous or granular material, and 70 percent was found in the soluble fraction. The Cm concentration initially associated with cell nuclei was high but had diminished to 20 percent of the 2 hr concentration by 20 days post injection (PI). The subcellular distribution of Cm in the liver of a dog which had received the same dose and was terminated because of severe liver damage was studied at 384 days PI. The liver weighed 130 g and contained approximately 30 percent of the injected Cm. In contrast, a normal liver weighs 280 g and at 2 hr PI contains approximately 40 percent of the injected dose. The subcellular distribution of Cm in this severely damaged liver differed from the pattern observed at earlier times after injection. The relative concentration of Cm in the cytosol was doubled; it was higher in the nuclei-debris fraction; and it was lower in the mitochondrial and lysosomal fractions when compared to earlier times

  1. 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

  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. Archaeal ammonia oxidizers respond to soil factors at smaller spatial scales than the overall archaeal community does in a high Arctic polar oasis.

    Science.gov (United States)

    Banerjee, Samiran; Kennedy, Nabla; Richardson, Alan E; Egger, Keith N; Siciliano, Steven D

    2016-06-01

    Archaea are ubiquitous and highly abundant in Arctic soils. Because of their oligotrophic nature, archaea play an important role in biogeochemical processes in nutrient-limited Arctic soils. With the existing knowledge of high archaeal abundance and functional potential in Arctic soils, this study employed terminal restriction fragment length polymorphism (t-RFLP) profiling and geostatistical analysis to explore spatial dependency and edaphic determinants of the overall archaeal (ARC) and ammonia-oxidizing archaeal (AOA) communities in a high Arctic polar oasis soil. ARC communities were spatially dependent at the 2-5 m scale (P diversity indices of both ARC and AOA communities showed high spatial dependency along the landscape and resembled scaling of edaphic factors. The spatial link between archaeal community structure and soil resources found in this study has implications for predictive understanding of archaea-driven processes in polar oases. PMID:27045904

  6. 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

  7. 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

  8. 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).

  9. Adaptations of proteins to cellular and subcellular pH

    OpenAIRE

    Garcia-Moreno, Bertrand

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69

  10. 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.

  11. 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.

  12. A re-evaluation of the archaeal membrane lipid biosynthetic pathway.

    Science.gov (United States)

    Villanueva, Laura; Damsté, Jaap S Sinninghe; Schouten, Stefan

    2014-06-01

    Archaea produce unique membrane lipids in which isoprenoid alkyl chains are bound to glycerol moieties via ether linkages. As cultured representatives of the Archaea have become increasingly available throughout the past decade, archaeal genomic and membrane lipid-composition data have also become available. In this Analysis article, we compare the amino acid sequences of the key enzymes of the archaeal ether-lipid biosynthesis pathway and critically evaluate past studies on the biochemical functions of these enzymes. We propose an alternative archaeal lipid biosynthetic pathway that is based on a 'multiple-key, multiple-lock' mechanism. PMID:24801941

  13. 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

  14. 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.

  15. 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

  16. 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.

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

    International Nuclear Information System (INIS)

    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

  18. Drivers of archaeal ammonia-oxidizing communities in soil

    Directory of Open Access Journals (Sweden)

    KaterynaZhalnina

    2012-06-01

    Full Text Available Soil ammonia-oxidizing archaea (AOA are highly abundant and play an important role in the nitrogen cycle. In addition, AOA have a significant impact on soil quality. AOA may cause nitrogen loss from soils, and the nitrate produced by AOA can lead to ground and surface water contamination, water eutrophication, and soil subsidence. The ammonia-oxidizing archaea discovered to date are classified in the phylum Thaumarchaeota. Only a few archaeal genomes are available in databases. As a result, AOA genes are not well annotated, and it is difficult to mine and identify archaeal genes within metagenomic libraries. Nevertheless, 16S rRNA and comparative analysis of ammonia monooxygenase sequences show that soils can vary greatly in the relative abundance of AOA. In some soils, AOA can comprise more than 10% of the total prokaryotic community. In other soils, AOA comprise less than 0.5% of the community. Many approaches have been used to measure the abundance and diversity of this group including DGGE, T-RFLP, q-PCR, and DNA sequencing. AOA have been studied across different soil types and various ecosystems from the Antarctic dry valleys to the tropical forests of South America to the soils near Mount Everest. Different studies have identified multiple soil factors that trigger the abundance of AOA. These factors include pH, concentration of available ammonia, organic matter content, moisture content, nitrogen content, clay content, as well as other triggers. Land use management appears to have a major effect on the abundance of AOA in soil, which may be the result of nitrogen fertilizer used in agricultural soils. This review summarizes the published results on this topic and suggests future work that will increase our understanding of how soil management and edaphoclimatic factors influence AOA.

  19. 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.

  20. 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...

  1. 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

  2. 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....... We used terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and clone libraries to characterize the soils' archaeal community composition to gain a better understanding of relationships between peat properties and land use, respectively, and CH4 dynamics. Samples were taken...... at three different depths and at four different seasons. Archaeal community composition varied considerably between the three peatlands and, to a certain degree, also with peat depth, but seemed to be quite stable at individual sampling depths throughout the year. Archaeal community composition was mainly...

  3. 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

  4. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

    Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

    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 benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. PMID:26356766

  5. 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.

  6. 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.

  7. 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...

  8. 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...

  9. 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...

  10. Adaptations of proteins to cellular and subcellular pH.

    Science.gov (United States)

    Garcia-Moreno, Bertrand

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69. PMID:20017887

  11. Cholinesterase of skeletal muscle and its subcellular components.

    OpenAIRE

    Fujii,Masafumi; Namba, Tatsuji

    1982-01-01

    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 frac...

  12. 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.

  13. Sequence Analysis and Comparative Study of the Protein Subunits of Archaeal RNase P

    Directory of Open Access Journals (Sweden)

    Manoj P. Samanta

    2016-04-01

    Full Text Available RNase P, a ribozyme-based ribonucleoprotein (RNP complex that catalyzes tRNA 5′-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes, we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs.

  14. Sequence Analysis and Comparative Study of the Protein Subunits of Archaeal RNase P.

    Science.gov (United States)

    Samanta, Manoj P; Lai, Stella M; Daniels, Charles J; Gopalan, Venkat

    2016-01-01

    RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5'-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs. PMID:27104580

  15. Sequence Analysis and Comparative Study of the Protein Subunits of Archaeal RNase P

    Science.gov (United States)

    Samanta, Manoj P.; Lai, Stella M.; Daniels, Charles J.; Gopalan, Venkat

    2016-01-01

    RNase P, a ribozyme-based ribonucleoprotein (RNP) complex that catalyzes tRNA 5′-maturation, is ubiquitous in all domains of life, but the evolution of its protein components (RNase P proteins, RPPs) is not well understood. Archaeal RPPs may provide clues on how the complex evolved from an ancient ribozyme to an RNP with multiple archaeal and eukaryotic (homologous) RPPs, which are unrelated to the single bacterial RPP. Here, we analyzed the sequence and structure of archaeal RPPs from over 600 available genomes. All five RPPs are found in eight archaeal phyla, suggesting that these RPPs arose early in archaeal evolutionary history. The putative ancestral genomic loci of archaeal RPPs include genes encoding several members of ribosome, exosome, and proteasome complexes, which may indicate coevolution/coordinate regulation of RNase P with other core cellular machineries. Despite being ancient, RPPs generally lack sequence conservation compared to other universal proteins. By analyzing the relative frequency of residues at every position in the context of the high-resolution structures of each of the RPPs (either alone or as functional binary complexes), we suggest residues for mutational analysis that may help uncover structure-function relationships in RPPs. PMID:27104580

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. A Meta-Analysis of the Bacterial and Archaeal Diversity Observed in Wetland Soils

    Directory of Open Access Journals (Sweden)

    Xiaofei Lv

    2014-01-01

    Full Text Available This study examined the bacterial and archaeal diversity from a worldwide range of wetlands soils and sediments using a meta-analysis approach. All available 16S rRNA gene sequences recovered from wetlands in public databases were retrieved. In November 2012, a total of 12677 bacterial and 1747 archaeal sequences were collected in GenBank. All the bacterial sequences were assigned into 6383 operational taxonomic units (OTUs 0.03, representing 31 known bacterial phyla, predominant with Proteobacteria (2791 OTUs, Bacteroidetes (868 OTUs, Acidobacteria (731 OTUs, Firmicutes (540 OTUs, and Actinobacteria (418 OTUs. The genus Flavobacterium (11.6% of bacterial sequences was the dominate bacteria in wetlands, followed by Gp1, Nitrosospira, and Nitrosomonas. Archaeal sequences were assigned to 521 OTUs from phyla Euryarchaeota and Crenarchaeota. The dominating archaeal genera were Fervidicoccus and Methanosaeta. Rarefaction analysis indicated that approximately 40% of bacterial and 83% of archaeal diversity in wetland soils and sediments have been presented. Our results should be significant for well-understanding the microbial diversity involved in worldwide wetlands.

  1. 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).

  2. Subcellular site and nature of intracellular cadmium in plants

    International Nuclear Information System (INIS)

    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 109CdCl2 or 112CdSO4 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 109Cd 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

  3. 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

  4. 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.

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

    Science.gov (United States)

    Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

    2015-09-01

    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. PMID:26484255

  6. 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

  7. 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

  8. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten; Dalgaard, Jacob; Lykke-Andersen, Jens; Phan, Hoa T.N.; Trevisanato, Siro; Østergaard, Laust; Larsen, Niels; Leffers, Henrik

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows is a...

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Archaeal communities in boreal forest tree rhizospheres respond to changing soil temperatures.

    Science.gov (United States)

    Bomberg, Malin; Münster, Uwe; Pumpanen, Jukka; Ilvesniemi, Hannu; Heinonsalo, Jussi

    2011-07-01

    Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7-11.5°C (night-day temperature), 12-16°C, and 16-22°C, of which 12-16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the

  16. 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.

  17. Differential response of archaeal groups to land use change in an acidic red soil.

    Science.gov (United States)

    Shen, Ju-Pei; Cao, Peng; Hu, Hang-Wei; He, Ji-Zheng

    2013-09-01

    Land use management, one of the most important aspects of anthropogenic disturbance to terrestrial ecosystems, has exerted overriding impacts on soil biogeochemical cycling and inhabitant microorganisms. However, the knowledge concerning response of different archaeal groups to long-term land use changes is still limited in terrestrial environments. Here we used quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE) approaches to investigate the response of archaeal communities to four different land use practices, i.e. cropland, pine forest, restoration land and degradation land. qPCR analyses showed that expression of the archaeal amoA gene responds more sensitively to changes of land use. In particular, we observed, occurring at significantly lower numbers of archaeal amoA genes in degradation land samples, while the abundance of total archaea and Group 1.1c based on 16S rRNA gene copy numbers remained constant among the different treatments examined. Soil nitrate content is significantly correlated with archaeal amoA gene abundance, but not their bacterial counterparts. The percentage of archaea among total prokaryote communities increases with increasing depth, but has no significant relationship with total carbon, total nitrogen or pH. Soil pH was significantly correlated with total bacterial abundance. Based on results from PCR-DGGE, three land use practices (i.e. cropland, pine forest, restoration land) showed distinct dominant bands, which were mostly affiliated with Group 1.1a. Degradation land, however, was dominated by sequences belonging to Group 1.1c. Results from this study suggest that community structure of ammonia oxidizing archaea were significantly impacted by land use practices. PMID:23774250

  18. 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...

  19. 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.

  20. 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...

  1. Convolutional LSTM Networks for Subcellular Localization of Proteins

    OpenAIRE

    Nielsen, Henrik; Sønderby, Søren Kaae; Sønderby, Casper Kaae; Winther, Ole

    2015-01-01

    Machine learning is widely used to analyze biological sequence data. Non-sequential models such as SVMs or feed-forward neural networks are often used although they have no natural way of handling sequences of varying length. Recurrent neural networks such as the long short term memory (LSTM) model on the other hand are designed to handle sequences. In this study we demonstrate that LSTM networks predict the subcellular location of proteins given only the protein sequence with high accuracy (...

  2. 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

  3. Discriminative Motif Finding for Predicting Protein Subcellular Localization

    OpenAIRE

    Lin, Tien-ho; Murphy, Robert F.; Bar-Joseph, Ziv

    2011-01-01

    Many methods have been described to predict the subcellular location of proteins from sequence information. However, most of these methods either rely on global sequence properties or use a set of known protein targeting motifs to predict protein localization. Here we develop and test a novel method that identifies potential targeting motifs using a discriminative approach based on hidden Markov models (discriminative HMMs). These models search for motifs that are present in a compartment but...

  4. 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.

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

    OpenAIRE

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

    2014-01-01

    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 ...

  6. 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

  7. 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

  8. 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...

  9. Gene Acquisitions from Bacteria at the Origins of Major Archaeal Clades Are Vastly Overestimated

    Science.gov (United States)

    Groussin, Mathieu; Boussau, Bastien; Szöllõsi, Gergely; Eme, Laura; Gouy, Manolo; Brochier-Armanet, Céline; Daubin, Vincent

    2016-01-01

    In a recent article, Nelson-Sathi et al. (NS) report that the origins of major archaeal lineages (MAL) correspond to massive group-specific gene acquisitions via HGT from bacteria (Nelson-Sathi et al. 2015. Origins of major archaeal clades correspond to gene acquisitions from bacteria. Nature 517(7532):77-80.). If correct, this would have fundamental implications for the process of diversification in microbes. However, a reexamination of these data and results shows that the methodology used by NS systematically inflates the number of genes acquired at the root of each MAL, and incorrectly assumes bacterial origins for these genes. A reanalysis of their data with appropriate phylogenetic models accounting for the dynamics of gene gain and loss between lineages supports the continuous acquisition of genes over long periods in the evolution of Archaea. PMID:26541173

  10. 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

  11. 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...

  12. 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...

  13. 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

  14. 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...

  15. 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...

  16. 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...

  17. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endon...... of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes....

  18. 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.

  19. 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. PMID:24837280

  20. 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 ...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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) ...

  6. 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...

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Image analysis of the movement of sub-cellular structures

    Czech Academy of Sciences Publication Activity Database

    Matula, Pe.; Ondřej, Vladan; Kozubek, Stanislav

    Brno : Masarykova univerzita v Brně, 2004 - (Kozubek, S.; Kozubek, M.), s. 62-65 ISBN 80-210-3560-9. [Biophysics of the Genome. Brno (CZ), 12.10.2004-13.10.2004] R&D Projects: GA ČR GA202/04/0907; GA AV ČR IBS5004010; GA AV ČR IAA5004306 Institutional research plan: CEZ:AV0Z5004920 Keywords : sub-cellular structures * image analysis * graph theory Subject RIV: BO - Biophysics

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Gene ontology based transfer learning for protein subcellular localization

    Directory of Open Access Journals (Sweden)

    Zhou Shuigeng

    2011-02-01

    Full Text Available Abstract Background Prediction of protein subcellular localization generally involves many complex factors, and using only one or two aspects of data information may not tell the true story. For this reason, some recent predictive models are deliberately designed to integrate multiple heterogeneous data sources for exploiting multi-aspect protein feature information. Gene ontology, hereinafter referred to as GO, uses a controlled vocabulary to depict biological molecules or gene products in terms of biological process, molecular function and cellular component. With the rapid expansion of annotated protein sequences, gene ontology has become a general protein feature that can be used to construct predictive models in computational biology. Existing models generally either concatenated the GO terms into a flat binary vector or applied majority-vote based ensemble learning for protein subcellular localization, both of which can not estimate the individual discriminative abilities of the three aspects of gene ontology. Results In this paper, we propose a Gene Ontology Based Transfer Learning Model (GO-TLM for large-scale protein subcellular localization. The model transfers the signature-based homologous GO terms to the target proteins, and further constructs a reliable learning system to reduce the adverse affect of the potential false GO terms that are resulted from evolutionary divergence. We derive three GO kernels from the three aspects of gene ontology to measure the GO similarity of two proteins, and derive two other spectrum kernels to measure the similarity of two protein sequences. We use simple non-parametric cross validation to explicitly weigh the discriminative abilities of the five kernels, such that the time & space computational complexities are greatly reduced when compared to the complicated semi-definite programming and semi-indefinite linear programming. The five kernels are then linearly merged into one single kernel for

  18. 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. PMID:27169490

  19. 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

  20. 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

  1. Archaeal community composition affects the function of anaerobic co-digesters in response to organic overload

    International Nuclear Information System (INIS)

    Highlights: ► Two types of methanogens are necessary to respond successfully to perturbation. ► Diversity of methanogens correlates with the VFA concentration and methane yield. ► Aggregates indicate tight spatial relationship between minerals and microorganisms. - Abstract: Microbial community diversity in two thermophilic laboratory-scale and three full-scale anaerobic co-digesters was analysed by genetic profiling based on PCR-amplified partial 16S rRNA genes. In parallel operated laboratory reactors a stepwise increase of the organic loading rate (OLR) resulted in a decrease of methane production and an accumulation of volatile fatty acids (VFAs). However, almost threefold different OLRs were necessary to inhibit the gas production in the reactors. During stable reactor performance, no significant differences in the bacterial community structures were detected, except for in the archaeal communities. Sequencing of archaeal PCR products revealed a dominance of the acetoclastic methanogen Methanosarcina thermophila, while hydrogenotrophic methanogens were of minor importance and differed additionally in their abundance between reactors. As a consequence of the perturbation, changes in bacterial and archaeal populations were observed. After organic overload, hydrogenotrophic methanogens (Methanospirillum hungatei and Methanoculleus receptaculi) became more dominant, especially in the reactor attributed by a higher OLR capacity. In addition, aggregates composed of mineral and organic layers formed during organic overload and indicated tight spatial relationships between minerals and microbial processes that may support de-acidification processes in over-acidified sludge. Comparative analyses of mesophilic stationary phase full-scale reactors additionally indicated a correlation between the diversity of methanogens and the VFA concentration combined with the methane yield. This study demonstrates that the coexistence of two types of methanogens, i

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Archaeal Distribution in Moonmilk Deposits from Alpine Caves and Their Ecophysiological Potential.

    Science.gov (United States)

    Reitschuler, Christoph; Spötl, Christoph; Hofmann, Katrin; Wagner, Andreas O; Illmer, Paul

    2016-04-01

    (Alpine) caves are, in general, windows into the Earth's subsurface. Frequently occurring structures in caves such as moonmilk (secondary calcite deposits) offer the opportunity to study intraterrestrial microbial communities, adapted to oligotrophic and cold conditions. This is an important research field regarding the dimensions of subsurface systems and cold regions on Earth. On a methodological level, moonmilk deposits from 11 caves in the Austrian Alps were collected aseptically and investigated using a molecular (qPCR and DGGE sequencing-based) methodology in order to study the occurrence, abundance, and diversity of the prevailing native Archaea community. Furthermore, these Archaea were enriched in complex media and studied regarding their physiology, with a media selection targeting different physiological requirements, e.g. methanogenesis and ammonia oxidation. The investigation of the environmental samples showed that all moonmilk deposits were characterized by the presence of the same few habitat-specific archaeal species, showing high abundances and constituting about 50 % of the total microbial communities. The largest fraction of these Archaea was ammonia-oxidizing Thaumarchaeota, while another abundant group was very distantly related to extremophilic Euryarchaeota (Moonmilk Archaea). The archaeal community showed a depth- and oxygen-dependent stratification. Archaea were much more abundant (around 80 %), compared to bacteria, in the actively forming surface part of moonmilk deposits, decreasing to about 5 % down to the bedrock. Via extensive cultivation efforts, it was possible to enrich the enigmatic Moonmilk Archaea and also AOA significantly above the level of bacteria. The most expedient prerequisites for cultivating Moonmilk Archaea were a cold temperature, oligotrophic conditions, short incubation times, a moonmilk surface inoculum, the application of erythromycin, and anaerobic (microaerophilic) conditions. On a physiological level, it

  7. 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...

  8. Quantification of asymmetric microtubule nucleation at sub-cellular structures

    Science.gov (United States)

    Zhu, Xiaodong; Kaverina, Irina

    2012-01-01

    Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in non-differentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centrosome (microtubule organizing center, MTOC) and then reorganized into the asymmetric array. We have recently identified the Golgi complex as an additional MTOC that asymmetrically nucleates MTs toward one side of the cell. Methods used for alternative MTOC identification include microtubule re-growth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescence labeled MT +TIP binding proteins in living cells. These approaches can be used for quantification of MT nucleation sites at diverse sub-cellular structures. PMID:21773933

  9. Cellular and subcellular distribution of BSH in human glioblastoma multiforme

    International Nuclear Information System (INIS)

    The cellular and subcellular distribution of mercaptoundecahydrododecaborate (BSH) in seven glioblastoma multiforme tissue sections of six patients having received BSH prior to surgery was investigated by light, fluorescence and electron microscopy. With use of specific antibodies against BSH its localization could be found in tissue sections predominantly (approx. 90%) in the cytoplasm of GFAP-positive cells of all but one patient. The latter was significantly younger (33 years in contrast of 46-71 (mean 60) years). In none of the tissue sections BSH could be found to a significant amount in the cell nuclei. In contrast, electron microscopy studies show BSH as well associated with the cell membrane as with the chromatin in the nucleus. (author)

  10. 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

  11. 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...

  12. 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 ...

  13. Archaeal diversity in a Fe-As rich acid mine drainage at Carnoules (France)

    OpenAIRE

    Bruneel, Odile; Pascault, N.; Egal, M; Bancon-Montigny, C.; Goni-urriza, M. S.; Elbaz Poulichet, F.; Personne, J. C.; Duran, R.

    2008-01-01

    The acid waters (pH = 2.73-3.4) that originate from the Carnoules mine tailings (France) are known for their very high concentrations of As (up to 10,000 mg l(-1)) and Fe (up to 20,000 mg l(-1)). To analyze the composition of the archaeal community, (their temporal variation inside the tailing and spatial variations all along the Reigous Creek, which drains the site), seven 16S rRNA gene libraries were constructed. Clone analysis revealed that all the sequences were affiliated to the phylum E...

  14. Free Energy Simulations of a GTPase: GTP and GDP Binding to Archaeal Initiation Factor 2

    OpenAIRE

    Satpati, Priyadarshi; Clavaguéra, Carine; Ohanessian, Gilles; Simonson, Thomas

    2011-01-01

    Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, “ON” conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, “OFF” conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free ene...

  15. 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 ...

  16. A reported archaeal mechanosensitive channel is a structural homolog of MarR-like transcriptional regulators

    OpenAIRE

    Liu, Zhenfeng; Walton, Troy A; Rees, Douglas C.

    2010-01-01

    Several archaeal mechanosensitive (MS) channels have been reported, including one from Thermoplasma volcanium designated MscTV. Here, we report the crystal structure of MscTV at 1.6-Å resolution. Unexpectedly, MscTV was found to be a water-soluble protein exhibiting a winged helix-turn-helix (wHTH) motif, which is the signature of the MarR (multiple antibiotic resistance regulator) family of transcriptional regulators. A cell-based osmotic downshock functional assay demonstrated that MscTV wa...

  17. 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.

  18. 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

  19. Cellular and subcellular localization of Marlin-1 in the brain

    Directory of Open Access Journals (Sweden)

    Luján Rafael

    2009-04-01

    Full Text Available Abstract Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  20. 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.

  1. 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.

  2. 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

  3. Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Chang, Ho-Won; Kim, Kyoung-Ho; Nam, Young-Do; Roh, Seong Woon; Kim, Min-Soo; Jeon, Che Ok; Oh, Hee-Mock; Bae, Jin-Woo

    2008-08-15

    Kimchi is a traditional Korean food that is fermented from vegetables such as Chinese cabbage and radish. Many bacteria are involved in kimchi fermentation and lactic acid bacteria are known to perform significant roles. Although kimchi fermentation presents a range of environmental conditions that could support many different archaea and yeasts, their molecular diversity within this process has not been studied. Here, we use PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 26S rRNA genes, to characterize bacterial, archaeal and yeast dynamics during various types of kimchi fermentation. The DGGE analysis of archaea expressed a change of DGGE banding patterns during kimchi fermentation, however, no significant change was observed in the yeast DGGE banding patterns during kimchi fermentation. No significant difference was indicated in the archaeal DGGE profile among different types of kimchi. In the case of yeasts, the clusters linked to the manufacturing corporation. Haloarchaea such as Halococcus spp., Natronococcus spp., Natrialba spp. and Haloterrigena spp., were detected as the predominant archaea and Lodderomyces spp., Trichosporon spp., Candida spp., Saccharomyces spp., Pichia spp., Sporisorium spp. and Kluyveromyces spp. were the most common yeasts. PMID:18562030

  4. Phylogenomic Dating-The Relative Antiquity of Archaeal Metabolic and Physiological Traits

    Science.gov (United States)

    Blank, Carrine E.

    2009-03-01

    Ancestral trait reconstruction was used to identify the relative ancestry of metabolic and physiological traits in the archaeal domain of life. First, well-resolved phylogenetic trees were inferred with multiple gene sequences obtained from whole genome sequences. Next, metabolic and physiological traits were coded into characters, and ancestral state reconstruction was used to identify ancient and derived traits. Traits inferred to be ancient included sulfur reduction, methanogenesis, and hydrogen oxidation. By using the articulation of the “oxygen age constraint,” several other traits were inferred to have arisen at or after 2.32 Ga: aerobic respiration, nitrate reduction, sulfate reduction, thiosulfate reduction, sulfur oxidation, and sulfide oxidation. Complex organic metabolism appeared to be nearly as ancient as autotrophy. Hyperthermophily was ancestral, while hyperacidophily and extreme halophily likely arose after 2.32 Ga. The ancestral euryarchaeote was inferred to have been a hyperthermophilic marine methanogen that lived in a deep-sea hydrothermal vent. In contrast, the ancestral crenarchaeote was most likely a hyperthermophilic sulfur reducer that lived in a slightly acidic terrestrial environment, perhaps a fumarole. Cross-colonization of these habitats may not have occurred until after 2.32 Ga, which suggests that both archaeal lineages exhibited niche specialization on early Earth for a protracted period of time.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. [Bacterial and archaeal diversity in surface sediment from the south slope of the South China Sea].

    Science.gov (United States)

    Li, Tao; Wang, Peng; Wang, Pinxian

    2008-03-01

    Diversity of bacteria and archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rDNA. Sample analysed was from IMAGES (International Marine Past Global Change Study) 147 at site of the south slope of the South China Sea. DNA was amplified from samples at the surface layer of core MD05-2896. Phylogenetic analysis of clone libraries showed a wide variety of uncultured bacteria and archeae. The most abundant bacterial sequences (phylotypes) corresponded to the Proteobacteria, followed by the Planctomycete, Acidobacteria and candidate division OP10. Phylotypes ascribing to Deferrobacteres, Verrucomicrobia, Spirochaetes and candidate division clades of OP3, OP11, OP8 and TM6 were also identified. Archaeal 16S rDNA sequences were within phylums of Crenarchaeota and Euryarchaeota, respectively. The majority of archaeal phylotypes were Marine Benthic Group B (MBGB), Marine Crenarchaeotic Group I (MG I), Marine Benthic Group D (MBGD) and South African Gold Mine Euryarchaeotic Group (SAGMEG). Additional sequences grouped with the C3, Methanobacteriales and Novel Euryarchaeotic Group (NEG). These results indicate that bacteria and archaea are abundant and diversified in surface environment of subseafloor sediments. PMID:18479058

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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; Rachel, Reinhard; Garrett, Roger A; Prangishvili, David

    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...

  19. 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.

  20. Subcellular localization of the antidepressant-sensitive norepinephrine transporter

    Directory of Open Access Journals (Sweden)

    Winder Danny G

    2009-06-01

    Full Text Available Abstract Background Reuptake of synaptic norepinephrine (NE via the antidepressant-sensitive NE transporter (NET supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons. Results We elucidate NET localization in brain and superior cervical ganglion (SCG neurons, aided by a new NET monoclonal antibody, subcellular immunoisolation techniques and quantitative immunofluorescence approaches. We present evidence that axonal NET extensively colocalizes with syntaxin 1A, and to a limited degree with SCAMP2 and synaptophysin. Intracellular NET in SCG axons and boutons also quantitatively segregates from the vesicular monoamine transporter 2 (VMAT2, findings corroborated by organelle isolation studies. At the surface of SCG boutons, NET resides in both lipid raft and non-lipid raft subdomains and colocalizes with syntaxin 1A. Conclusion Our findings support the hypothesis that SCG NET is segregated prior to transport from the cell body from proteins comprising large dense core vesicles. Once localized to presynaptic boutons, NET does not recycle via VMAT2-positive, small dense core vesicles. Finally, once NET reaches presynaptic plasma membranes, the transporter localizes to syntaxin 1A-rich plasma membrane domains, with a portion found in cholera toxin-demarcated lipid rafts. Our findings indicate that activity-dependent insertion of NET into the SCG plasma membrane derives from vesicles distinct from those that deliver NE. Moreover, NET is localized in presynaptic membranes in a manner that can take advantage of regulatory processes targeting lipid raft subdomains.

  1. 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.

  2. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

    Science.gov (United States)

    Serag, Maged F; Braeckmans, Kevin; Habuchi, Satoshi; Kaji, Noritada; Bianco, Alberto; Baba, Yoshinobu

    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. PMID:23170917

  3. Subcellular localization of hepatitis E virus (HEV) replicase

    International Nuclear Information System (INIS)

    Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of ∼ 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication

  4. 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

  5. Determination of arsenic in subcellular fractions of rat liver by ENAA

    International Nuclear Information System (INIS)

    The concentration of arsenic in subcellular fractions of rat liver is determined by differential centrifugation combined with epithermal neutron activation analysis (ENAA). The analytical quality has been verified by analysis of some standard reference materials. The results show that the distribution of As in subcellular fractions of rat liver cells is not homogeneous. The highest concentration of As is found in microsome, while the lowest content is found in nuclei for arsenic poisoned rat. The lowest content of As is noted in mitochondria of contrast rats. The differences of As contents in subcellular fractions of lives cells between arsenic poisoned and normal rats are compared by means of t-test and exceedingly significant differences are found (P<0.001). In As poisoned rat, the As content in microsome is significantly higher than that in other subcellular fractions (P<0.05), except lysosome

  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. 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...

  8. Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

    OpenAIRE

    Greiser, Maura; Kerfant, Benoît-Gilles; Williams, George S. B.; Voigt, Niels; Harks, Erik; Dibb, Katharine M.; Giese, Anne; Meszaros, Janos; Verheule, Sander; Ravens, Ursula; Allessie, Maurits A.; Gammie, James S.; Van Der Velden, Jolanda; Lederer, W. Jonathan; Dobrev, Dobromir

    2014-01-01

    Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ wa...

  9. 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...

  10. Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

    OpenAIRE

    Masedunskas, Andrius; Porat-Shliom, Natalie; Tora, Muhibullah; Milberg, Oleg; Weigert, Roberto

    2013-01-01

    Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most o...

  11. 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

  12. 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

  13. 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...

  14. Structure-Based Engineering of Lithium-Transport Capacity in an Archaeal Sodium-Calcium Exchanger.

    Science.gov (United States)

    Refaeli, Bosmat; Giladi, Moshe; Hiller, Reuben; Khananshvili, Daniel

    2016-03-29

    Members of the Ca(2+)/cation exchanger superfamily (Ca(2+)/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca(2+), Na(+), H(+), K(+), and Li(+). The archaeal Na(+)/Ca(2+) exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na(+), whereas the mitochondrial ortholog (NCLX) can transport either Li(+) or Na(+) in exchange with Ca(2+). Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na(+) or Li(+), similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins. PMID:26958982

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

    Science.gov (United States)

    Pineda De Castro, Luis Felipe; Dopson, Mark

    2016-01-01

    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. PMID:27167213

  16. 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

  17. 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.

  18. Structure of the acidianus filamentous virus 3 and comparative genomics of related archaeal lipothrixviruses

    DEFF Research Database (Denmark)

    Vestergaard, Gisle Alberg; Aramayo, Ricardo; Basta, Tamara;

    2008-01-01

    Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The...... structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an...... high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be...

  19. Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life

    DEFF Research Database (Denmark)

    Prangishvili, D.; Garrett, R. A.; Koonin, E.

    2006-01-01

    the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure of...... accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable...... homologs. Here we describe a re-analysis of the proteins encoded by archaeal viruses, with an emphasis on comparative genomics of the unique viruses of Crenarchaeota. Detailed examination of conserved domains and motifs uncovered a significant number of previously unnoticed homologous relationships among...

  20. Contrasting spatial patterns and ecological attributes of soil bacterial and archaeal taxa across a landscape.

    Science.gov (United States)

    Constancias, Florentin; Saby, Nicolas P A; Terrat, Sébastien; Dequiedt, Samuel; Horrigue, Wallid; Nowak, Virginie; Guillemin, Jean-Philippe; Biju-Duval, Luc; Chemidlin Prévost-Bouré, Nicolas; Ranjard, Lionel

    2015-06-01

    Even though recent studies have clarified the influence and hierarchy of environmental filters on bacterial community structure, those constraining bacterial populations variations remain unclear. In consequence, our ability to understand to ecological attributes of soil bacteria and to predict microbial community response to environmental stress is therefore limited. Here, we characterized the bacterial community composition and the various bacterial taxonomic groups constituting the community across an agricultural landscape of 12 km(2) , by using a 215 × 215 m systematic grid representing 278 sites to precisely decipher their spatial distribution and drivers at this scale. The bacterial and Archaeal community composition was characterized by applying 16S rRNA gene pyrosequencing directly to soil DNA from samples. Geostatistics tools were used to reveal the heterogeneous distribution of bacterial composition at this scale. Soil physical parameters and land management explained a significant amount of variation, suggesting that environmental selection is the major process shaping bacterial composition. All taxa systematically displayed also a heterogeneous and particular distribution patterns. Different relative influences of soil characteristics, land use and space were observed, depending on the taxa, implying that selection and spatial processes might be differentially but not exclusively involved for each bacterial phylum. Soil pH was a major factor determining the distribution of most of the bacterial taxa and especially the most important factor explaining the spatial patterns of α-Proteobacteria and Planctomycetes. Soil texture, organic carbon content and quality were more specific to a few number of taxa (e.g., β-Proteobacteria and Chlorobi). Land management also influenced the distribution of bacterial taxa across the landscape and revealed different type of response to cropping intensity (positive, negative, neutral or hump-backed relationships

  1. Microbial community structure analysis of a benzoate-degrading halophilic archaeal enrichment.

    Science.gov (United States)

    Dalvi, Sonal; Youssef, Noha H; Fathepure, Babu Z

    2016-05-01

    A benzoate-degrading archaeal enrichment was developed using sediment samples from Rozel Point at Great Salt Lake, UT. The enrichment degraded benzoate as the sole carbon source at salinity ranging from 2.0 to 5.0 M NaCl with highest rate of degradation observed at 4.0 M. The enrichment was also tested for its ability to grow on other aromatic compounds such as 4-hydroxybenzoic acid (4-HBA), gentisic acid, protocatechuic acid (PCA), catechol, benzene and toluene as the sole sources of carbon and energy. Of these, the culture only utilized 4-HBA as the carbon source. To determine the initial steps in benzoate degradation pathway, a survey of ring-oxidizing and ring-cleaving genes was performed using degenerate PCR primers. Results showed the presence of 4-hydroxybenzoate 3-monooxygenase (4-HBMO) and protocatechuate 3, 4-dioxygenase (3,4-PCA) genes suggesting that the archaeal enrichment might degrade benzoate to 4-HBA that is further converted to PCA by 4-HBMO and, thus, formed PCA would undergo ring-cleavage by 3,4-PCA to form intermediates that enter the Krebs cycle. Small subunit rRNA gene-based diversity survey revealed that the enrichment consisted entirely of class Halobacteria members belonging to the genera Halopenitus, Halosarcina, Natronomonas, Halosimplex, Halorubrum, Salinarchaeum and Haloterrigena. Of these, Halopenitus was the dominant group accounting for almost 91 % of the total sequences suggesting their potential role in degrading oxygenated aromatic compounds at extreme salinity. PMID:26995683

  2. 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.

  3. 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

  4. Rhesus macaque MHC class I molecules show differential subcellular localizations.

    Science.gov (United States)

    Rosner, Cornelia; Kruse, Philip H; Lübke, Torben; Walter, Lutz

    2010-03-01

    The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding alpha1 and alpha2 domains, suggesting failure of peptide binding is responsible for retaining 'intracellular' Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes. PMID:20151120

  5. 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

  6. 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.

  7. 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 ...

  8. 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...

  9. 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...

  10. 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.

  11. 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

  12. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    Science.gov (United States)

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states. PMID:26005782

  13. 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

  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. 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...

  16. 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 LethJørgensen

    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.

  17. 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

  18. 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 ...

  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 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.

  1. 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

  2. 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.

  3. Identification of GH15 Family Thermophilic Archaeal Trehalases That Function within a Narrow Acidic-pH Range.

    Science.gov (United States)

    Sakaguchi, Masayoshi; Shimodaira, Satoru; Ishida, Shin-Nosuke; Amemiya, Miko; Honda, Shotaro; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2015-08-01

    Two glucoamylase-like genes, TVN1315 and Ta0286, from the archaea Thermoplasma volcanium and T. acidophilum, respectively, were expressed in Escherichia coli. The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α)6 barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display Km values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants (Ki) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism. PMID:25979886

  4. 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.

  5. 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

  6. 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.

  7. 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

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

    International Nuclear Information System (INIS)

    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: CaCl2-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 CaCl2 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 assessed

  9. 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

  10. 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.

  11. Study of Se subcellular distribution in human tissues by neutron activation analysis

    International Nuclear Information System (INIS)

    Subcellular distribution of selenium in certain important human tissues is studied by neutron activation analysis combined with differential centrifugation method. The standard reference materials, bovine liver NIST 1577a and horse kidney IAEA H8, are used to validate the analytical quality. The experimental results show that the slenium levels in various individuals are significantly different. Body selenium is highly enriched in human kidney. Different subcellular selenium distribution patterns are also found in different tissues. The selenium levels in various organelles are sequenced as mitochondria >nuclei>cytosol>lysosome, microsome in liver; nuclei>mitochondria>cytosol>lysosome, microsome in kidney; and mitochondria, nuclei>cytosol, lysosome, microsome in heart

  12. Concentration of 17 Elements in Subcellular Fractions of Beef Heart Tissue Determined by Neutron Activation Analysis

    International Nuclear Information System (INIS)

    Subcellular fractions of beef heart tissue are investigated, by means of neutron activation analysis, with respect to their concentration of 17 different elements. A recently developed ion-exchange technique combined with gamma spectrometry is used. The homogeneity of the subcellular fractions is examined electron microscopically. The following elements are determined: As, Ba, Br, Cas Co, Cs, Cu, Fe, Hg, La, Mo, P, Rb, Se, Sm, W and Zn. The determination of Ag, Au, Cd, Ce, Cr, Sb and Sc is omitted, in view of contamination. Reproducible and characteristic patterns of distribution are obtained for all elements studied

  13. 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.

  14. Archaeal remains dominate marine organic matter from the early Albian oceanic anoxic event 1b

    DEFF Research Database (Denmark)

    Kuypers, M.M.M.; Blokker, P.; Hopmans, E.C.;

    2002-01-01

    The sources for both soluble and insoluble organic matter of the early Albian (∼112 Myr) oceanic anoxic event (OAE) 1b black shales of the Ocean Drilling Program (ODP) site 1049C (North Atlantic Ocean off the coast of Florida) and the Ravel section of the Southeast France Basin (SEFB) were...... C/C ratios was used to estimate that up to ∼40% of the organic matter of the SEFB and up to ∼80% of the organic matter of ODP site 1049C preserved in the black shales is derived from archaea. Furthermore, it is shown that, even though there are apparent similarities (high organic carbon (OC) content......, distinct lamination, C-enrichment of OC) between the black shales of OAE1b and the Cenomanian/Turonian (∼94 Myr) OAE, the origin of the organic matter (archaeal versus phytoplanktonic) and causes for C-enrichment of OC are completely different. © 2002 Elsevier Science B.V. All rights reserved....

  15. 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

  16. 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

  17. Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase.

    Science.gov (United States)

    Carbone, Vincenzo; Schofield, Linley R; Zhang, Yanli; Sang, Carrie; Dey, Debjit; Hannus, Ingegerd M; Martin, William F; Sutherland-Smith, Andrew J; Ronimus, Ron S

    2015-08-28

    One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn(2+) cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn(2+) cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH. PMID:26175150

  18. 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

  19. 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

  20. 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

  1. 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

  2. Comparison of bacterial and archaeal communities in depth-resolved zones in an LNAPL body.

    Science.gov (United States)

    Irianni-Renno, Maria; Akhbari, Daria; Olson, Mitchell R; Byrne, Adam P; Lefèvre, Emilie; Zimbron, Julio; Lyverse, Mark; Sale, Thomas C; De Long, Susan K

    2016-04-01

    Advances in our understanding of the microbial ecology at sites impacted by light non-aqueous phase liquids (LNAPLs) are needed to drive development of optimized bioremediation technologies, support longevity models, and develop culture-independent molecular tools. In this study, depth-resolved characterization of geochemical parameters and microbial communities was conducted for a shallow hydrocarbon-impacted aquifer. Four distinct zones were identified based on microbial community structure and geochemical data: (i) an aerobic, low-contaminant mass zone at the top of the vadose zone; (ii) a moderate to high-contaminant mass, low-oxygen to anaerobic transition zone in the middle of the vadose zone; (iii) an anaerobic, high-contaminant mass zone spanning the bottom of the vadose zone and saturated zone; and (iv) an anaerobic, low-contaminant mass zone below the LNAPL body. Evidence suggested that hydrocarbon degradation is mediated by syntrophic fermenters and methanogens in zone III. Upward flux of methane likely contributes to promoting anaerobic conditions in zone II by limiting downward flux of oxygen as methane and oxygen fronts converge at the top of this zone. Observed sulfate gradients and microbial communities suggested that sulfate reduction and methanogenesis both contribute to hydrocarbon degradation in zone IV. Pyrosequencing revealed that Syntrophus- and Methanosaeta-related species dominate bacterial and archaeal communities, respectively, in the LNAPL body below the water table. Observed phylotypes were linked with in situ anaerobic hydrocarbon degradation in LNAPL-impacted soils. PMID:26691516

  3. 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

  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.

    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.

  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.

    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

  6. 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.

  7. 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...

  8. Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

    International Nuclear Information System (INIS)

    To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-[1-3H]galactose or [3H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [3H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid

  9. 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.

  10. High resolution intravital imaging of subcellular structures of mouse abdominal organs using a microstage device.

    Science.gov (United States)

    Cao, Liqin; Kobayakawa, Satoru; Yoshiki, Atsushi; Abe, Kuniya

    2012-01-01

    Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox. PMID:22479464

  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. Subcellular binding of 239Pu in the liver of selected species of rodents

    International Nuclear Information System (INIS)

    The subcellular distribution of 239Pu in the liver of selected rodent species was investigated as well as the relation between 239Pu and the iron metabolism. The goal of the investigation was to find out why the liver discharge of 239Pu from the liver varies so much between species. (orig.)

  13. 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

  14. 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. PMID:24563838

  15. 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

  16. Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss)

    International Nuclear Information System (INIS)

    Highlights: Interactions of Cu, Cd and Zn were studied at the subcellular level in rainbow trout. Metals accumulated in the liver were predominantly metabolically active. Cd, Cu and Zn exhibited both competitive and cooperative interactions. The metal–metal interactions altered subcellular metals partitioning. - Abstract: Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing (μg/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67–83%; Cu, 68–79% and Zn, 60–76

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

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

    International Nuclear Information System (INIS)

    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

  3. Diversity of Archaeal Consortia in an Arsenic-Rich Hydrothermal System

    Science.gov (United States)

    Franks, M.; Bennett, P.; Omelon, C.; Engel, A.

    2008-12-01

    Characterizing microbial communities within their geochemical environment is essential to understanding microbial distribution and microbial adaptations to extreme physical and chemical conditions. The hydrothermal waters at El Tatio geyser field demonstrate extreme conditions, with water at local boiling (85°C), arsenic concentrations at 0.5 mM, and inorganic carbon concentrations as low as 0.02mM. Yet many of El Tatio's hundred plus hydrothermal features are associated with extensive microbial mat communities. Recent work has shown phylogenetic variation in the communities that correlates to variations in water chemistry between features. MPN analysis indicates variations in metabolic function between hydrothermal features, such as the ability of the community to fix nitrogen, and the presence of methanogens within the community. Methanogenic archaea, which are typical of hydrothermal environments, are found in very few of the sampled hydrothermal features at El Tatio. MPN enumeration shows that nonspecific microbial mat samples from sites with dissolved methane contain 106 cells of methanogenic archaea per gram while non-specific samples from sites lacking dissolved methane contain 100 cells per gram or less. An acetylene assay showed evidence for nitrogen fixation in a sample associated with methanogenesis, but microbial transformation of acetylene to ethylene did not occur in non-methanogenic sites. More specific sampling of microbial mats indicates that methanogenic archaea are dominated by microorganisms within the genus Methanospirillum and Methanobrevibacter. These microbes are associated with a number of unclassified archaea in the class Thermoplasmata Halobacteriales, and unclassifiec Crenarchaeota. In addition, preliminary results include an unclassified Thaumarchaeota clone, a member of the recently proposed third archaeal phylum Thaumarchaeota. Nonspecific microbial mat sample from a non- methanogenic site included only Crenarchaeal clones within the

  4. Tracing the Archaeal Origins of Eukaryotic Membrane-Trafficking System Building Blocks.

    Science.gov (United States)

    Klinger, Christen M; Spang, Anja; Dacks, Joel B; Ettema, Thijs J G

    2016-06-01

    In contrast to prokaryotes, eukaryotic cells are characterized by a complex set of internal membrane-bound compartments. A subset of these, and the protein machineries that move material between them, define the membrane-trafficking system (MTS), the emergence of which represents a landmark in eukaryotic evolution. Unlike mitochondria and plastids, MTS organelles have autogenous origins. Much of the MTS machinery is composed of building blocks, including small GTPase, coiled-coil, beta-propeller + alpha-solenoid, and longin domains. Despite the identification of prokaryotic proteins containing these domains, only few represent direct orthologues, leaving the origins and early evolution of the MTS poorly understood. Here, we present an in-depth analysis of MTS building block homologues in the composite genome of Lokiarchaeum, the recently discovered archaeal sister clade of eukaryotes, yielding several key insights. We identify two previously unreported Eukaryotic Signature Proteins; orthologues of the Gtr/Rag family GTPases, involved in target of rapamycin complex signaling, and of the RLC7 dynein component. We could not identify golgin or SNARE (coiled-coil) or beta-propeller + alpha-solenoid orthologues, nor typical MTS domain fusions, suggesting that these either were lost from Lokiarchaeum or emerged later in eukaryotic evolution. Furthermore, our phylogenetic analyses of lokiarchaeal GTPases support a split into Ras-like and Arf-like superfamilies, with different prokaryotic antecedents, before the advent of eukaryotes. While no GTPase activating proteins or exchange factors were identified, we show that Lokiarchaeum encodes numerous roadblock domain proteins and putative longin domain proteins, confirming the latter's origin from Archaea. Altogether, our study provides new insights into the emergence and early evolution of the eukaryotic membrane-trafficking system. PMID:26893300

  5. 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

  6. 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.

  7. 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

  8. 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...

  9. 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...

  10. 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.

  11. Free energy simulations of a GTPase: GTP and GDP binding to archaeal initiation factor 2.

    Science.gov (United States)

    Satpati, Priyadarshi; Clavaguéra, Carine; Ohanessian, Gilles; Simonson, Thomas

    2011-05-26

    Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force

  12. 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

  13. 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.

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

    DEFF Research Database (Denmark)

    Nielsen, Joachim; Ørtenblad, Niels

    2013-01-01

    and fatigability. Based on all these data, the available literature strongly indicates that the subcellular localization of glycogen has to be taken into consideration to fully understand and appreciate the role and regulation of glycogen metabolism and signaling in skeletal muscle. A full......Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electron microscopy images as roughly spherical particles (known as β-particles of 10-45 nm in diameter), which are distributed in distinct localizations within the myofibers and are physically associated...... 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...

  15. 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.

  16. 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

  17. 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.

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

    International Nuclear Information System (INIS)

    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.

  19. Emergent cell and tissue dynamics from subcellular modeling of active biomechanical processes

    International Nuclear Information System (INIS)

    Cells and the tissues they form are not passive material bodies. Cells change their behavior in response to external biochemical and biomechanical cues. Behavioral changes, such as morphological deformation, proliferation and migration, are striking in many multicellular processes such as morphogenesis, wound healing and cancer progression. Cell-based modeling of these phenomena requires algorithms that can capture active cell behavior and their emergent tissue-level phenotypes. In this paper, we report on extensions of the subcellular element model to model active biomechanical subcellular processes. These processes lead to emergent cell and tissue level phenotypes at larger scales, including (i) adaptive shape deformations in cells responding to slow stretching, (ii) viscous flow of embryonic tissues, and (iii) streaming patterns of chemotactic cells in epithelial-like sheets. In each case, we connect our simulation results to recent experiments

  20. 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.

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

    OpenAIRE

    1984-01-01

    Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determine...

  6. APSLAP: an adaptive boosting technique for predicting subcellular localization of apoptosis protein.

    Science.gov (United States)

    Saravanan, Vijayakumar; Lakshmi, P T V

    2013-12-01

    Apoptotic proteins play key roles in understanding the mechanism of programmed cell death. Knowledge about the subcellular localization of apoptotic protein is constructive in understanding the mechanism of programmed cell death, determining the functional characterization of the protein, screening candidates in drug design, and selecting protein for relevant studies. It is also proclaimed that the information required for determining the subcellular localization of protein resides in their corresponding amino acid sequence. In this work, a new biological feature, class pattern frequency of physiochemical descriptor, was effectively used in accordance with the amino acid composition, protein similarity measure, CTD (composition, translation, and distribution) of physiochemical descriptors, and sequence similarity to predict the subcellular localization of apoptosis protein. AdaBoost with the weak learner as Random-Forest was designed for the five modules and prediction is made based on the weighted voting system. Bench mark dataset of 317 apoptosis proteins were subjected to prediction by our system and the accuracy was found to be 100.0 and 92.4 %, and 90.1 % for self-consistency test, jack-knife test, and tenfold cross validation test respectively, which is 0.9 % higher than that of other existing methods. Beside this, the independent data (N151 and ZW98) set prediction resulted in the accuracy of 90.7 and 87.7 %, respectively. These results show that the protein feature represented by a combined feature vector along with AdaBoost algorithm holds well in effective prediction of subcellular localization of apoptosis proteins. The user friendly web interface "APSLAP" has been constructed, which is freely available at http://apslap.bicpu.edu.in and it is anticipated that this tool will play a significant role in determining the specific role of apoptosis proteins with reliability. PMID:23982307

  7. Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization.

    OpenAIRE

    Fleming, R.E.; Parkkila, S; Parkkila, A K; Rajaniemi, H; Waheed, A; Sly, W S

    1995-01-01

    Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-linked isozyme previously identified on the surface of renal tubular epithelium and certain populations of vascular endothelium. This report identifies the regional, cellular, and subcellular localization of CA IV in the rat gut. Northern blot and RT-PCR analyses demonstrated little CA IV expression in stomach or proximal small intestine, but abundant expression in distal small and large intestine. In contrast, CA II mRNA was abu...

  8. 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...

  9. 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...

  10. 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% ...

  11. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope

    OpenAIRE

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-ichi; Ashida, Hitoshi

    2009-01-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning f...

  12. 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 ...

  13. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold.

    OpenAIRE

    Tinglu, G; Ghosh, A.; Ghosh, B K

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles...

  14. *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 ...

  15. 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.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Xia Li

    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.

  18. 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

  19. 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)

  20. Tissue and subcellular distribution of zinc-65 in normal and zinc sulphate administered rats

    International Nuclear Information System (INIS)

    Uptake of 65Zn by various organs of normal and ZnSO4 administered rats has been studied. Except duodenum and blood all the organs show low 65Zn incorporation in treated rats. The lower 65Zn incorporation in the tissues of ZnSO4 treated rats is due to the active turnover of zinc in these tissues. The maximum activity of 65Zn is exhibited by skin, followed in decreasing order by liver, intestine, spleen, rectum, kidney, prostate, urinary bladder, stomach, pancreas, adrenals, trachea, lungs, heart, thymus, seminal vesicles, testis, cauda epididymis, caput epididymis, brain, and muscle. Since ZnSO4 administration enhanced the levels of inert zinc in liver and intestine it is likely that accumulated inert zinc in the tissues may inhibit the further incorporation of 65Zn in these tissues. At subcellular level, the cytosol fraction (11000 g supernatant fraction) exhibits the higher 65Zn activity, followed in decreasing order by 300 g fraction (cell membranes and cell debris), 900 g fraction (nuclei), 6000 g fraction (heavy mitochondria) and 1000 g fraction (light mitochondria). The 65Zn activity/mgprotein in 11000 g fraction (light mitochondria) is more than the 900 g fraction (nuclei) and 6000 g fraction (heavy mitochondria). Subcellular distribution pattern of 65Zn in liver, intestine, kidney and testis is similar. ZnSO4 administration does not change the tissue and subcellular distribution pattern of 65Zn. (author). 18 refs., 3 tables

  1. 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

  2. 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.

  3. 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.

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

    International Nuclear Information System (INIS)

    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.

  6. Uptake pathways and subcellular fractionation of Cd in the polychaete Nereis diversicolor.

    Science.gov (United States)

    Li, Lianzhen; Liu, Xiaoli; You, Liping; Zhang, Linbao; Zhao, Jianmin; Wu, Huifeng

    2012-01-01

    Polychaetes have often been utilized as indicator species to investigate the impacts of pollutants, such as heavy metals. The uptake of Cd by the polychaete Nereis diversicolor was determined at varying Ca concentrations and with pre-exposure to Ca ion channel blockers and metabolic inhibitors in simulated sea water over 1 week period. The supply of Ca in simulated sea water inhibited Cd uptake and increased Ca concentration in N. diversicolor after 10 μM Cd exposure. Pre-exposure to a Ca-channel blocker (Lanthanum) significantly inhibited Cd uptake, suggesting that the uptake of Cd was exerted at a Ca channel. N-ethylmaleimide, which specifically binds to sulfhydryl groups, inhibited Cd uptake at 10 μM, implying that the transport of Cd is carrier-mediated by proteins or other SH-containing compounds. Subcellular Cd distribution analysis showed that more than 60% of the total Cd associated with the cytosolic fraction. The presence of higher concentration of Ca in simulated sea water did not impact the proportional subcellular distribution of Cd in N. diversicolor. Nevertheless, the supply of Ca could significantly lower Cd concentration in cytosol and cellular debris. The present study provides evidence that Cd transport by N. diversicolor was mediated mainly through lanthanum- sensitive Ca ion channels and accumulated by SH-containing compounds. These results help to understand the uptake mechanism and subcellular distribution of Cd in polychaetes. PMID:21858512

  7. PathLocdb: a comprehensive database for the subcellular localization of metabolic pathways and its application to multiple localization analysis

    OpenAIRE

    Zhao Min; Qu Hong

    2010-01-01

    Abstract Background In eukaryotes, the cell is divided into several compartments enclosed by unitary membranes. Such compartmentalization is critical for cells to restrict different pathways to be carried out in different subcellular regions. The summary and classification of subcellular localizations of metabolic pathways are the first steps towards understanding their roles in spatial differentiation and the specialization of metabolic pathways in different organisms. Results Integrating th...

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

    OpenAIRE

    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, allantoicase and ureidoglycollate lyase were shown to be cytosolic. In addition the peroxisomes harboured catalase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase.

  9. Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells: FROM ENTRY OF EXTRACELLULAR PRECURSORS TO MITOCHONDRIAL NAD GENERATION*

    OpenAIRE

    Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

    2011-01-01

    NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribo...

  10. 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

  11. Geranylgeranyl reductase and ferredoxin from Methanosarcina acetivorans are required for the synthesis of fully reduced archaeal membrane lipid in Escherichia coli cells.

    Science.gov (United States)

    Isobe, Keisuke; Ogawa, Takuya; Hirose, Kana; Yokoi, Takeru; Yoshimura, Tohru; Hemmi, Hisashi

    2014-01-01

    Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme. PMID:24214941

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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...

  18. 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 crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and......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...... periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable...

  19. 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...

  20. Methanogenic pathway and archaeal communities in three different anoxic soils amended with rice straw and maize straw

    Directory of Open Access Journals (Sweden)

    RalfConrad

    2012-01-01

    Full Text Available Addition of straw is common practice in rice agriculture, but its effect on the path of microbial CH4 production and the microbial community involved is not well known. Since straw from rice (C3 plant and maize plants (C4 plant exhibit different δ13C values, we compared the effect of these straw types using anoxic rice field soils from Italy and China, and also a soil from Thailand that had previously not been flooded. The temporal patterns of production of CH4 and its major substrates H2 and acetate, were slightly different between rice straw and maize straw. Addition of methyl fluoride, an inhibitor of aceticlastic methanogenesis, resulted in partial inhibition of acetate consumption and CH4 production. The δ13C of the accumulated CH4 and acetate reflected the different δ13C values of rice straw versus maize straw. However, the relative contribution of hydrogenotrophic methanogenesis to total CH4 production exhibited a similar temporal change when scaled to CH4 production irrespectively of whether rice straw or maize straw was applied. The composition of the methanogenic archaeal communities was characterized by terminal restriction fragment length polymorphism (T-RFLP analysis and was quantified by quantitative PCR (qPCR targeting archaeal 16S rRNA genes or methanogenic mcrA genes.. The size of the methanogenic communities generally increased during incubation with straw, but the straw type had little effect. Instead, differences were found between the soils, with Methanosarcinaceae and Methanobacteriales dominating straw decomposition in Italian soil, Methanosarcinaceae, Methanocellales, and Methanobacteriale in China soil, and Methanosarcinaceae and Methanocellales in Thailand soil. The experiments showed that methanogenic degradation in different soils involved different methanogenic population dynamics. However, the path of CH4 production was hardly different between degradation of rice straw versus maize straw and was also similar for

  1. Structure of Mth11/Mth Rpp29, an essential protein subunit of archaeal and eukaryotic RNase P.

    Science.gov (United States)

    Boomershine, William P; McElroy, Craig A; Tsai, Hsin-Yue; Wilson, Ross C; Gopalan, Venkat; Foster, Mark P

    2003-12-23

    We have determined the solution structure of Mth11 (Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs in all three domains of life. In eubacteria, this enzyme is made up of two subunits: a large RNA ( approximately 120 kDa) responsible for mediating catalysis, and a small protein cofactor ( approximately 15 kDa) that modulates substrate recognition and is required for efficient in vivo catalysis. In contrast, multiple proteins are associated with eukaryotic and archaeal RNase P, and these proteins exhibit no recognizable homology to the conserved bacterial protein subunit. In reconstitution experiments with recombinantly expressed and purified protein subunits, we found that Mth Rpp29, a homolog of the Rpp29 protein subunit from eukaryotic RNase P, is an essential protein component of the archaeal holoenzyme. Consistent with its role in mediating protein-RNA interactions, we report that Mth Rpp29 is a member of the oligonucleotide/oligosaccharide binding fold family. In addition to a structured beta-barrel core, it possesses unstructured N- and C-terminal extensions bearing several highly conserved amino acid residues. To identify possible RNA contacts in the protein-RNA complex, we examined the interaction of the 11-kDa protein with the full 100-kDa Mth RNA subunit by using NMR chemical shift perturbation. Our findings represent a critical step toward a structural model of the RNase P holoenzyme from archaebacteria and higher organisms. PMID:14673079

  2. 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.

  3. Buccal swabbing as a noninvasive method to determine bacterial, archaeal, and eukaryotic microbial community structures in the rumen.

    Science.gov (United States)

    Kittelmann, Sandra; Kirk, Michelle R; Jonker, Arjan; McCulloch, Alan; Janssen, Peter H

    2015-11-01

    Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants. PMID:26276109

  4. Distribution of ether lipids and composition of the archaeal community in terrestrial geothermal springs: impact of environmental variables.

    Science.gov (United States)

    Xie, Wei; Zhang, Chuanlun L; Wang, Jinxiang; Chen, Yufei; Zhu, Yuanqing; de la Torre, José R; Dong, Hailiang; Hartnett, Hilairy E; Hedlund, Brian P; Klotz, Martin G

    2015-05-01

    Archaea can respond to changes in the environment by altering the composition of their membrane lipids, for example, by modification of the abundance and composition of glycerol dialkyl glycerol tetraethers (GDGTs). Here, we investigated the abundance and proportions of polar GDGTs (P-GDGTs) and core GDGTs (C-GDGTs) sampled in different seasons from Tengchong hot springs (Yunnan, China), which encompassed a pH range of 2.5-10.1 and a temperature range of 43.7-93.6°C. The phylogenetic composition of the archaeal community (reanalysed from published work) divided the Archaea in spring sediment samples into three major groups that corresponded with spring pH: acidic, circumneutral and alkaline. Cluster analysis showed correlation between spring pH and the composition of P- and C-GDGTs and archaeal 16S rRNA genes, indicating an intimate link between resident Archaea and the distribution of P- and C-GDGTs in Tengchong hot springs. The distribution of GDGTs in Tengchong springs was also significantly affected by temperature; however, the relationship was weaker than with pH. Analysis of published datasets including samples from Tibet, Yellowstone and the US Great Basin hot springs revealed a similar relationship between pH and GDGT content. Specifically, low pH springs had higher concentrations of GDGTs with high numbers of cyclopentyl rings than neutral and alkaline springs, which is consistent with the predominance of high cyclopentyl ring-characterized Sulfolobales and Thermoplasmatales present in some of the low pH springs. Our study suggests that the resident Archaea in these hot springs are acclimated if not adapted to low pH by their genetic capacity to effect the packing density of their membranes by increasing cyclopentyl rings in GDGTs at the rank of community. PMID:25142282

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. The subcellular localization of IGFBP5 affects its cell growth and migration functions in breast cancer

    Directory of Open Access Journals (Sweden)

    Sahin Aysegul

    2009-04-01

    Full Text Available Abstract Background Insulin-like growth factor binding protein 5 (IGFBP5 has been shown to be associated with breast cancer metastasis in clinical marker studies. However, a major difficulty in understanding how IGFBP5 functions in this capacity is the paradoxical observation that ectopic overexpression of IGFBP5 in breast cancer cell lines results in suppressed cellular proliferation. In cancer tissues, IGFBP5 resides mainly in the cytoplasm; however, in transfected cells, IGFBP5 is mainly located in the nucleus. We hypothesized that subcellular localization of IGFBP5 affects its functions in host cells. Methods To test this hypothesis, we generated wild-type and mutant IGFBP5 expression constructs. The mutation occurs within the nuclear localization sequence (NLS of the protein and is generated by site-directed mutagenesis using the wild-type IGFBP5 expression construct as a template. Next, we transfected each expression construct into MDA-MB-435 breast cancer cells to establish stable clones overexpressing either wild-type or mutant IGFBP5. Results Functional analysis revealed that cells overexpressing wild-type IGFBP5 had significantly lower cell growth rate and motility than the vector-transfected cells, whereas cells overexpressing mutant IGFBP5 demonstrated a significantly higher ability to proliferate and migrate. To illustrate the subcellular localization of the proteins, we generated wild-type and mutant IGFBP5-pDsRed fluorescence fusion constructs. Fluorescence microscopy imaging revealed that mutation of the NLS in IGFBP5 switched the accumulation of IGFBP5 from the nucleus to the cytoplasm of the protein. Conclusion Together, these findings imply that the mutant form of IGFBP5 increases proliferation and motility of breast cancer cells and that mutation of the NLS in IGFBP5 results in localization of IGFBP5 in the cytoplasm, suggesting that subcellular localization of IGFBP5 affects its cell growth and migration functions in the

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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 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......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...

  16. 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...

  17. 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.

  18. Hematoporphyrin derivative induced photodamage to brain tumor cells: Alterations in subcellular membranes

    Science.gov (United States)

    Sreenivasan, Rajesh; Joshi, Preeti G.; Joshi, Nanda B.

    1997-01-01

    Photoinduced structural and functional changes were studied in the subcellular membranes isolated from HpD treated cells. Changes in the limiting anisotropy of lipid specific probes 1,6,Diphenyl-1,3,5,hexatriene (DPH) and 1-(4-Trimethyl ammonium 1,6 diphenyl)-1,3,5,hexatriene toulene sulphonate (TMA-DPH) incorporated into the membrane were used to assess the structural alterations while changes in the activity of the marker enzymes were used to assess the functional alterations. Our results suggest that damage to the endoplasmic reticulum may play an important role in the photosensitization of brain tumor cells.

  19. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate; Breinholt Bekker-Jensen, Dorte; Secher, Anna; Skovgaard, Tine; Kelstrup, Christian; Dmytriyev, Anatoliy; Choudhary, Chuna Ram; Lundby, Carsten; Olsen, Jesper Velgaard

    2012-01-01

    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...... 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...

  20. 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.

  1. 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...

  2. 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 ...

  3. 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.

  4. 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...

  5. An improved procedure for subcellular spatial alignment during live-cell CLEM.

    Directory of Open Access Journals (Sweden)

    Benjamin S Padman

    Full Text Available Live-cell correlative light and electron microscopy (CLEM offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

  6. Tissue and subcellular localizations of 3H-cyclosporine A in mice

    International Nuclear Information System (INIS)

    The tissue and subcellular localizations of 3H-cyclosporine A after administration to mice were determined with whole-body autoradiography and scintillation counting of lipid extracts of tissues and subcellular fractions. The radioactivity was widely distributed in the body and the pattern of distribution after oral or parenteral administration was the same, except that tissue levels were generatlly lower after oral administration. Pretreatment of the animals with a diet containing cyclosporine A for 30 days before the injection of radioactive cyclosporine A did not change the pattern of distribution substantially. No significant radioactivity was found in the central nervous system, except for the choroidal plexus and the area postrema region of the brain. In pregnant mice no passage of radioactivity from the placentas to fetuses was observed after a single injection. 3H-cyclosporine A and/or its metabolites showed a high affinity for the lympho-myeloid tissues, with a marked long-term retention in bone marrow and lymph nodes. There was massive excretion in the intestinal tract after parenteral administration, and the liver, bile, pancreas and salivary glands contained high levels of radioactivity. In the kidney radioactivity was confined to the outer zone of the outer kidney medulla. In liver homogenates no quantitatively significant binding of 3H-cyclosporine A and/or its metabolites to cellular molecules such as proteins, DNA, phospho- or neutral lipids was found. After lipid extraction with organic solvents, almost all radioactivity was recovered in the organic phase. (author)

  7. Classification of Subcellular Phenotype Images by Decision Templates for Classifier Ensemble

    Science.gov (United States)

    Zhang, Bailing

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. An automatic, reliable and efficient prediction system for protein subcellular localization is needed for large-scale genome analysis. The automated cell phenotype image classification problem is an interesting "bioimage informatics" application. It 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, three well-known texture feature extraction methods including local binary patterns (LBP), Gabor filtering and Gray Level Coocurrence Matrix (GLCM) have been applied to cell phenotype images and the multiple layer perceptron (MLP) method has been used to classify cell phenotype image. After classification of the extracted features, decision-templates ensemble algorithm (DT) is used to combine base classifiers built on the different feature sets. Different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. For the HeLa cells, the human classification error rate on this task is of 17% as reported in previous publications. We obtain with our method an error rate of 4.8%.

  8. 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.)

  9. Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.

    Directory of Open Access Journals (Sweden)

    Zhang Yi

    2010-09-01

    Full Text Available Abstract Background Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency. Results Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake ( Conclusions Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.

  10. Spreading the news: subcellular and organellar reactive oxygen species production and signalling.

    Science.gov (United States)

    Mignolet-Spruyt, Lorin; Xu, Enjun; Idänheimo, Niina; Hoeberichts, Frank A; Mühlenbock, Per; Brosché, Mikael; Van Breusegem, Frank; Kangasjärvi, Jaakko

    2016-06-01

    As plants are sessile organisms that have to attune their physiology and morphology continuously to varying environmental challenges in order to survive and reproduce, they have evolved complex and integrated environment-cell, cell-cell, and cell-organelle signalling circuits that regulate and trigger the required adjustments (such as alteration of gene expression). Although reactive oxygen species (ROS) are essential components of this network, their pathways are not yet completely unravelled. In addition to the intrinsic chemical properties that define the array of interaction partners, mobility, and stability, ROS signalling specificity is obtained via the spatiotemporal control of production and scavenging at different organellar and subcellular locations (e.g. chloroplasts, mitochondria, peroxisomes, and apoplast). Furthermore, these cellular compartments may crosstalk to relay and further fine-tune the ROS message. Hence, plant cells might locally and systemically react upon environmental or developmental challenges by generating spatiotemporally controlled dosages of certain ROS types, each with specific chemical properties and interaction targets, that are influenced by interorganellar communication and by the subcellular location and distribution of the involved organelles, to trigger the suitable acclimation responses in association with other well-established cellular signalling components (e.g. reactive nitrogen species, phytohormones, and calcium ions). Further characterization of this comprehensive ROS signalling matrix may result in the identification of new targets and key regulators of ROS signalling, which might be excellent candidates for engineering or breeding stress-tolerant plants. PMID:26976816

  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. Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

    Science.gov (United States)

    Greiser, Maura; Kerfant, Benoît-Gilles; Williams, George S.B.; Voigt, Niels; Harks, Erik; Dibb, Katharine M.; Giese, Anne; Meszaros, Janos; Verheule, Sander; Ravens, Ursula; Allessie, Maurits A.; Gammie, James S.; van der Velden, Jolanda; Lederer, W. Jonathan; Dobrev, Dobromir; Schotten, Ulrich

    2014-01-01

    Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia. PMID:25329692

  13. Subcellular targeting and dynamic regulation of PTEN: Implications for neuronal cells and neurological disorders

    Directory of Open Access Journals (Sweden)

    Ivo Lieberam

    2014-04-01

    Full Text Available PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum, the mitochondria or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  14. Analysis of potato virus X replicase and TGBp3 subcellular locations

    International Nuclear Information System (INIS)

    Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

  15. 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.

  16. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L

    International Nuclear Information System (INIS)

    Phytolacca americana L. (pokeweed) is a promising species for Cd phytoextraction with large biomass and fast growth rate. To further understand the mechanisms involved in Cd tolerance and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in pokeweed. Subcellular fractionation of Cd-containing tissues indicated that both in root and leaves, the majority of the element was located in soluble fraction and cell walls. Meanwhile, Cd taken up by pokeweed existed in different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 80% ethanol in roots, followed by 1 M NaCl, d-H2O and 2% HAc, while in leaves and stems, most of the Cd was extracted by 1 M NaCl, and the subdominant amount of Cd was extracted by 80% ethanol. It could be suggested that Cd compartmentation with organo-ligands in vacuole or integrated with pectates and proteins in cell wall might be responsible for the adaptation of pokeweed to Cd stress.

  17. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    Science.gov (United States)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  18. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

    Science.gov (United States)

    Tanz, Sandra K; Castleden, Ian; Hooper, Cornelia M; Small, Ian; Millar, A Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1-Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  19. 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)

  20. β-amylase in developing apple fruits: activities, amounts and subcellular localization

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Dapeng; (张大鹏); WANG; Yongzhang(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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Steen Redeker, E. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium)]. E-mail: erik.steenredeker@ua.ac.be; Campenhout, K. van [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Bervoets, L. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Reijnders, H. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); Blust, R. [Ecophysiology, Biochemistry and Toxicology Group, Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium)

    2007-07-15

    We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed. - The internal metal speciation in prey items is not clearly linked to the actual assimilation in predators.

  8. Subcellular distribution of Cd in the aquatic oligochaete Tubifex tubifex, implications for trophic availability and toxicity

    International Nuclear Information System (INIS)

    We studied the compartmentalization of cadmium and zinc in the oligochaete Tubifex tubifex. The subcellular distribution was followed over time and levels of metallothionein-like proteins were measured. The impact of the speciation on the trophic transfer was studied by calculating the assimilation efficiencies of metals from Tubificidae fed to carp. It was found that carp were able to assimilate 9.8% of the cadmium. The expected assimilated amount of cadmium, based on the subcellular fractions which are thought to be trophically available, is however 72%. The zinc assimilation results suggest that the debris fraction is at least partially available to predators. Differential centrifugation techniques provide information about the tissue compartmentalization in aquatic organisms but it is not straightforward to directly link internal speciation in prey items to the actual assimilation in the predator. The possible impact that the compartmentalization of cadmium in T. tubifex will have on the toxicity to the organism is also discussed. - The internal metal speciation in prey items is not clearly linked to the actual assimilation in predators

  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. Bioaccumulation and subcellular partitioning of zinc in rainbow trout (Oncorhynchus mykiss): Cross-talk between waterborne and dietary uptake

    International Nuclear Information System (INIS)

    Zinc homeostasis was studied at the tissue and gill subcellular levels in rainbow trout (Oncorhynchus mykiss) following waterborne and dietary exposures, singly and in combination. Juvenile rainbow trout were exposed to 150 or 600 μg l-1 waterborne Zn, 1500 or 4500 μg g-1 dietary Zn, and a combination of 150 μg l-1 waterborne and 1500 μg g-1 dietary Zn for 40 days. Accumulation of Zn in tissues and gill subcellular fractions was measured. At the tissue level, the carcass acted as the main Zn depot containing 84-90% of whole body Zn burden whereas the gill held 4-6%. At the subcellular level, the majority of gill Zn was bioavailable with the estimated metabolically active pool being 81-90%. Interestingly, the nuclei-cellular debris fraction bound the highest amount (40%) of the gill Zn burden. There was low partitioning of Zn into the detoxified pool (10-19%) suggesting that sequestration and chelation are not major mechanisms of cellular Zn homeostasis in rainbow trout. Further, the subcellular partitioning of Zn did not conform to the spill-over model of metal toxicity because Zn binding was indiscriminate irrespective of exposure concentration and duration. The contribution of the branchial and gastrointestinal uptake pathways to Zn accumulation depended on the tissue. Specifically, in plasma, blood cells, and gill, uptake from water was dominant whereas both pathways appeared to contribute equally to Zn accumulation in the carcass. Subcellularly, additive uptake from the two pathways was observed in the heat-stable proteins (HSP) fraction. Toxicologically, Zn exposure caused minimal adverse effects manifested by a transitory inhibition of protein synthesis in gills in the waterborne exposure. Overall, subcellular fractionation appears to have value in the quest for a better understanding of Zn homeostasis and interactions between branchial and gastrointestinal uptake pathways

  11. 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...

  12. 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...

  13. 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.

  14. 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

  15. Novel archaeal macrocyclic diether core membrane lipids in a methane-derived carbonate crust from a mud volcano in the Sorokin Trough, NE Black Sea

    Directory of Open Access Journals (Sweden)

    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.

  16. 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

  17. 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

  18. 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

  19. Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reimann, Julia; Esser, Dominik; Orell, Alvaro; Amman, Fabian; Pham, Trong Khoa; Noirel, Josselin; Lindås, Ann-Christin; Bernander, Rolf; Wright, Phillip C; Siebers, Bettina; Albers, Sonja-Verena

    2013-12-01

    In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius. PMID:24078887

  20. Physiology, phylogeny and in situ evidence for bacterial and archaeal nitrifiers in the marine sponge Aplysina aerophoba.

    Science.gov (United States)

    Bayer, Kristina; Schmitt, Susanne; Hentschel, Ute

    2008-11-01

    The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges. PMID:18363713

  1. Quantification of bacterial and archaeal symbionts in high and low microbial abundance sponges using real-time PCR.

    Science.gov (United States)

    Bayer, Kristina; Kamke, Janine; Hentschel, Ute

    2014-09-01

    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. PMID:24942664

  2. 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.

  3. 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.

  4. 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.

  5. Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer

    International Nuclear Information System (INIS)

    TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P43212 or P41212, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Å resolution. 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 P43212 or P41212, 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 (VM) 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

  6. Temporal and Spatial Coexistence of Archaeal and Bacterial amoA Genes and Gene Transcripts in Lake Lucerne

    Directory of Open Access Journals (Sweden)

    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.

  7. 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

  8. 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.

  9. Structure and diversity of bacterial, eukaryotic and archaeal communities in glacial cryoconite holes from the Arctic and the Antarctic.

    Science.gov (United States)

    Cameron, Karen A; Hodson, Andrew J; Osborn, A Mark

    2012-11-01

    The cryosphere presents some of the most challenging conditions for life on earth. Nevertheless, (micro)biota survive in a range of niches in glacial systems, including water-filled depressions on glacial surfaces termed cryoconite holes (centimetre to metre in diameter and up to 0.5 m deep) that contain dark granular material (cryoconite). In this study, the structure of bacterial and eukaryotic cryoconite communities from ten different locations in the Arctic and Antarctica was compared using T-RFLP analysis of rRNA genes. Community structure varied with geography, with greatest differences seen between communities from the Arctic and the Antarctic. DNA sequencing of rRNA genes revealed considerable diversity, with individual cryoconite hole communities containing between six and eight bacterial phyla and five and eight eukaryotic 'first-rank' taxa and including both bacterial and eukaryotic photoautotrophs. Bacterial Firmicutes and Deltaproteobacteria and Epsilonproteobacteria, eukaryotic Rhizaria, Haptophyta, Choanomonada and Centroheliozoa, and archaea were identified for the first time in cryoconite ecosystems. Archaea were only found within Antarctic locations, with the majority of sequences (77%) related to members of the Thaumarchaeota. In conclusion, this research has revealed that Antarctic and Arctic cryoconite holes harbour geographically distinct highly diverse communities and has identified hitherto unknown bacterial, eukaryotic and archaeal taxa, therein. PMID:22168226

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Taurine effects on 45Ca2+ transport in retinal subcellular fractions

    International Nuclear Information System (INIS)

    The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1-5 min in a Krebs-bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role of taurine in the regulation of calcium fluxes in the retina. (Auth.)

  15. 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.

  16. 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

  17. 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

  18. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    International Nuclear Information System (INIS)

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table

  19. 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

  20. 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

  1. Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPres level disparity

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    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.

  2. Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

    Science.gov (United States)

    Zhong, Zhenhui; Norvienyeku, Justice; Yu, Jie; Chen, Meilian; Cai, Renli; Hong, Yonghe; Chen, Liqiong; Zhang, Dongmei; Wang, Baohua; Zhou, Jie; Lu, Guodong; Chen, Xiaofeng; Wang, Zonghua

    2015-10-01

    The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue. PMID:26318870

  3. Cadmium in three marine phytoplankton: Accumulation, subcellular fate and thiol induction

    International Nuclear Information System (INIS)

    We explored the possible mechanisms leading to differential Cd sensitivity in three marine phytoplankton (the diatom Thalassiosira pseudonana, the dinoflagellate Prorocentrum minimum and the green alga Chlorella autotrophica) based on their Cd accumulation, Cd subcellular distribution, and phytochelatin (PC) synthesis. The most sensitive species, T. pseudonana, generally exhibited the highest Cd body burden and organelle (org)-Cd concentration. C. autotrophica, the most tolerant species to Cd, had the smallest org-Cd accumulation, as well as a much higher percentage of cellular debris-Cd, which may play an important role in Cd detoxification. The dinoflagellate P. minimum, with a sensitivity between the diatoms and green algae, had a comparable Cd body burden but higher percentage of org-Cd than C. autotrophica. Although the induction of PCs was dependent on the species, the intracellular (intra)-Cd/PC-SH ratio showed a strong linear log-log relationship with [Cd2+], suggesting that this ratio could possibly be a biomarker for environmental [Cd2+] stress. With the increases of the intra-Cd/PC-SH ratio, these three species of phytoplankton exhibited clearly different patterns of growth inhibition, implying that the effectiveness of PCs as a detoxification pathway is dependent on the species. The lowest intra-Cd/PC-SH toxicity threshold for T. pseudonana implied its low PC-Cd capacity. Furthermore, the sudden slowdown of growth inhibition when the intra-Cd/PC-SH ratio reached 33 implied the launch of other detoxification pathway in C. autotrophica in order to alleviate Cd toxicity. Our study demonstrated that accumulation and subcellular distribution of Cd and PC synthesis can account for the inter-species differences in Cd sensitivity in marine phytoplankton.

  4. Subcellular trafficking of the amyloid precursor protein gene family and its pathogenic role in Alzheimer's disease.

    Science.gov (United States)

    Kins, Stefan; Lauther, Nadine; Szodorai, Anita; Beyreuther, Konrad

    2006-01-01

    Changes in the intracellular transport of amyloid precursor protein (APP) affect the extent to which APP is exposed to alpha- or beta-secretase in a common subcellular compartment and therefore directly influence the degree to which APP undergoes the amyloidogenic pathway leading to generation of beta-amyloid. As the presynaptic regions of neurons are thought to be the main source of beta-amyloid in the brain, attention has been focused on axonal APP trafficking. APP is transported along axons by a fast, kinesin-dependent anterograde transport mechanism. Despite the wealth of in vivo and in vitro data that have accumulated regarding the connection of APP to kinesin transport, it is not yet clear if APP is coupled to its specific motor protein via an intracellular interaction partner, such as the c-Jun N-terminal kinase-interacting protein, or by yet another unknown molecular mechanism. The cargo proteins that form a functional complex with APP are also unknown. Due to the long lifespan, and vast extent, of neurons, in particular axons, neurons are highly sensitive to changes in subcellular transport. Recent in vitro and in vivo studies have shown that variations in APP or tau affect mitochondrial and synaptic vesicle transport. Further, it was shown that this axonal dysfunction might lead to impaired synaptic plasticity, which is crucial for neuronal viability and function. Thus, changes in APP and tau expression may cause perturbed axonal transport and changes in APP processing, contributing to cognitive decline and neurodegeneration in Alzheimer's disease. PMID:17047360

  5. 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

  6. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    Science.gov (United States)

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  7. 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)

  8. Determination of ABA-binding proteins contents in subcellular fractions isolated from cotton seedlings using radioimmunoanalysis

    International Nuclear Information System (INIS)

    Full text: Knowledge of plants' hormone receptor sites is essential to understanding of the principles of phytohormone action in cells and tissues. The hormone abscisic acid (ABA) takes part in many important physiological processes of plants, including water balance and resistance to salt stress. The detection of salt tolerance in the early stages of ontogenesis is desirable for effective cultivation of cotton. Usually such characteristics are determined visually after genetic analysis of hybrids over several generations. This classic method of genetics requires a long time to grow several generations of cotton plants. In this connection we study ABA-binding protein contents in subcellular fractions isolated from seedlings of several kinds of cotton with different tolerance to salt stress. The contents of ABA-binding protein in nuclei and chloroplasts fractions isolated from cotton seedlings were determined using radioimmunoanalysis. The subcellular fractions were prepared by ultracentrifugation in 0,25 - 2,2 M sucrose gradient. ABA-binding protein was isolated from cotton seedlings by affinity chromatography. The antibodies against ABA-binding protein of cotton were developed in rabbits according standard protocols. Than the antibodies were labelled by radioisotope J125 according Greenwood et al. It was shown, that the nuclei and chloroplasts fractions isolated from cotton with high tolerance to salt stress contain ABA-binding protein up to 1,5-1,8 times more, than the same fractions from cotton with low tolerance to salt stress. So, the ABA-binding protein contents in cotton seedlings may be considered as a marker for screening of cotton kinds, which may potentially have high tolerance to salt stress

  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. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells.

    Science.gov (United States)

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. Movies of cellular and sub-cellular motion by digital holographic microscopy

    Directory of Open Access Journals (Sweden)

    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

  4. Ecological nanotoxicology: integrating nanomaterial hazard considerations across the subcellular, population, community, and ecosystems levels.

    Science.gov (United States)

    Holden, Patricia A; Nisbet, Roger M; Lenihan, Hunter S; Miller, Robert J; Cherr, Gary N; Schimel, Joshua P; Gardea-Torresdey, Jorge L

    2013-03-19

    Research into the health and environmental safety of nanotechnology has seriously lagged behind its emergence in industry. While humans have often adopted synthetic chemicals without considering ancillary consequences, the lessons learned from worldwide pollution should motivate making nanotechnology compatible with environmental concerns. Researchers and policymakers need to understand exposure and harm of engineered nanomaterials (ENMs), currently nanotechnology's main products, to influence the ENM industry toward sustainable growth. Yet, how should research proceed? Standard toxicity testing anchored in single-organism, dose-response characterizations does not adequately represent real-world exposure and receptor scenarios and their complexities. Our approach is different: it derives from ecology, the study of organisms' interactions with each other and their environments. Our approach involves the characterization of ENMs and the mechanistic assessment of their property-based effects. Using high throughput/content screening (HTS/HCS) with cells or environmentally-relevant organisms, we measure the effects of ENMs on a subcellular or population level. We then relate those effects to mechanisms within dynamic energy budget (DEB) models of growth and reproduction. We reconcile DEB model predictions with experimental data on organism and population responses. Finally, we use microcosm studies to measure the potential for community- or ecosystem-level effects by ENMs that are likely to be produced in large quantities and for which either HTS/HCS or DEB modeling suggest their potential to harm populations and ecosystems. Our approach accounts for ecological interactions across scales, from within organisms to whole ecosystems. Organismal ENM effects, if propagated through populations, can alter communities comprising multiple populations (e.g., plant, fish, bacteria) within food webs. Altered communities can change ecosystem services: processes that cycle carbon

  5. Spatial Variations in Archaeal Lipids of Surface Water and Core-Top Sediments in the South China Sea: Implications for Paleoclimate Studies

    Science.gov (United States)

    Wei, Y.; Wang, J.; Liu, J.; Dong, L.; Li, L.; Wang, H.; Wang, P.; Zhao, M.; Zhang, C.

    2011-12-01

    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 0.05±0.13 ng/L; 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 g/g, which are in 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 6-36.4% of total lipids (CL+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 depth shallower 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 the 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.

  6. 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.)

  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. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    OpenAIRE

    Amero, Carlos D; Boomershine, William P.; Xu, Yiren; Foster, Mark

    2008-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21...

  9. 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

  10. 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

  11. 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

  12. 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

  13. Comparative studies on the influence of some radioprotectors on the postirradiation hydrolytic function of subcellular fractions of pancreas

    International Nuclear Information System (INIS)

    Comparative studies on the influence of the two radioprotectors Fenchlorfos and cysteamine on the hydroproteolytic activity of pancreatic subcellular fractions of rats were carried out. The animals were irradiated with the single dose of 800 R of X-rays. Biochemical estimation of lipase, β-glucuronidase, acid phosphatase and catheptic (caseonolytic) activity were done 24 hours after exposition. The two radioprotective substances have a lot of common in their action against hydrolytic activity. They induce the decrease of some enzymes of subcellular fractions we checked both in control as well as in irradiated groups. The ambivalent influence of Trichlorfon on the lipase activity in granules and supernatant fraction seems to be of interest. (orig.)

  14. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types.

    Science.gov (United States)

    Dubois, Maïtée; Hare, Landis

    2009-03-01

    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. PMID:19110352

  15. Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types

    Energy Technology Data Exchange (ETDEWEB)

    Dubois, Maitee [Institut national de la recherche scientifique - Eau, Terre et Environnement, Universite du Quebec, Quebec City, Quebec, G1K 9A9 (Canada); Hare, Landis [Institut national de la recherche scientifique - Eau, Terre et Environnement, Universite du Quebec, Quebec City, Quebec, G1K 9A9 (Canada)], E-mail: landis@ete.inrs.ca

    2009-03-15

    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.

  16. A radiotracer study on the chronic effects and subcellular partitioning of metals in the Manila clam Ruditapes Philippinarum

    International Nuclear Information System (INIS)

    A laboratory microcosm study was employed to evaluate chronic physiological and histopathological effects of wate-rborne Ag to the Manila clam Ruditapes philippinarum. Additionally, radiotracers of gamma emitting isotopes of 109Cd, 51Cr, and 65Zn were used to study the metal bioaccumulation mechanisms and subcellular partitioning in the clam Ruditapes philippinarum. Bioaccumulation of Ag in the clam increased linearly with Ag concentration in seawater and exposure time. All the physiological parameters (e.g., filtration rate, scope for growth) and histopathological data had negative relationships with tissue Agconcentrations in the clams. The uptake rate of 3 metals increased in the order of Zn > Cd > Cr and had inverse relationship with salinity and with individual clam size (dry weight). The result suggest that subcellular partitioning of metals in the clams into different binding pools could be important factors determining Biologically Detoxified Metal (BDM), Trophically Available Metal (TAM) and Trophically Available Metal (TAM)

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Photoaffinity labelling of tobacco subcellular fractions with [32P]-azido-UDP-glucose

    International Nuclear Information System (INIS)

    Subcellular fractions from tobacco (N. rustica) leaves were photolabelled with the UDPG analog, [32P]-N3 UDPG. Two stromal polypeptides (Mr = 42 and 21 kD) were the major photolabelled chloroplast polypeptides. UDPG protected the 42 but not the 21 kD polypeptide against photoincorporation of [32P]-N3 UDPG. In a cytosol-enriched fraction, the major photolabelling polypeptides had Mr of 92, 50, 42, 30 and 17 kD. Photolabelling of the 42, 30, and 17 kD polypeptides was unaffected by UDPG, but UDPG blocked incorporation into the 92 and 50 kD polypeptides. In addition to photolabelled polypeptides, a polypeptide identified as phosphoglucomutase (PGM) was labelled in both the chloroplast and cytosol fraction. 32P-labelling of PGM was independent of UV irradiation, occurring via phosphoryl transfer from contaminating [32P]G-I-P. The plastid and cytosolic PGM isozymes had Mr of 69 and 62.8 kD, respectively, and both were labelled in a leaf extract

  2. Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

    International Nuclear Information System (INIS)

    Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions in the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species

  3. 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.

  4. 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

  5. Biochemical characterization and subcellular localization of the red kidney bean purple acid phosphatase

    International Nuclear Information System (INIS)

    Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the nonspecificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination

  6. 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. PMID:25451863

  7. Subcellular distribution of histone-degrading enzyme activities from rat liver

    International Nuclear Information System (INIS)

    Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. (orig.)

  8. 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.

  9. Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

    Institute of Scientific and Technical Information of China (English)

    QIU; Quansheng; (邱全胜); WANG; Zezhou(王泽宙); CAI; Qigui(蔡起贵); JIANG; Rongxi(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.

  10. 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

  11. Subcellular Clearance and Accumulation of Huntington Disease Protein: A Mini-Review

    Science.gov (United States)

    Zhao, Ting; Hong, Yan; Li, Xiao-Jiang; Li, Shi-Hua

    2016-01-01

    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 (UPS) 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 examining mHtt clearance and accumulation in different subcellular regions. PMID:27147961

  12. 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)

  13. 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

  14. 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.

  15. Incorporation of 15N into subcellular fractions and soluble proteins in rice seedlings

    International Nuclear Information System (INIS)

    15N incorporation into the subcellular and protein fractions of rice seedlings was investigated, when (15NH4)2SO4 was fed through culture solution, and the following was found. (1) Optical emission spectroscopic method is very useful for the 15N analysis of small amount of protein as much as separated by Sephadex G-200. (2) In the developed leaves, where nitrogen was balanced by continuous influx and efflux, 6 to 9 percent of total nitrogen was exchanged in a day by newly absorbed nitrogen, and the absorbed nitrogen was incorporated into chloroplast, 30,000 x g precipitate and soluble protein fractions by almost equal rates. (3) In the developing leaf, where the amount of nitrogen was increasing, newly absorbed and redistributed nitrogen almost equally contributed to the increase of total and protein nitrogen. (4) The speed of 15N incorporation into Fraction 1 Protein was the same in the developing leaf and slow in the developed leaves in comparison with other soluble proteins. (author)

  16. 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

  17. 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.

  18. 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.

  19. Subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

    International Nuclear Information System (INIS)

    Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes

  20. 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.

  1. Technical advance: Inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments.

    Science.gov (United States)

    Paschke, Stephan; Weidner, Astrid Franziska; Paust, Tobias; Marti, Othmar; Beil, Michael; Ben-Chetrit, Eldad

    2013-11-01

    Colchicine is an efficient drug for the management of inflammatory diseases, such as gouty arthritis and familial Mediterranean fever. It affects neutrophil activity by interfering with the formation of microtubules. To test the hypothesis that therapeutic concentrations of colchicine modulate the mechanical properties of these cells, we applied a combination of biophysical techniques (optical stretching and microrheology) to analyze cellular deformability. The contribution of the subcellular compartments to the regulation of cell mechanics was determined by fitting a multicomponent model of cellular viscoelasticity to time-dependent deformation curves. Neutrophils were found to be less deformable in response to 10 ng/ml colchicine. The model-based analysis of cellular deformation revealed a decrease in cytoplasmatic elasticity and a substantial increase in both elasticity and viscosity of the cell membrane compartment in response to colchicine. These results correlate with a reduced number of cytoplasmatic microtubules and an increase in subcortical actin filaments. The latter finding was confirmed by microrheology and fluorescence microscopy. Neutrophil migration through small pores requiring substantial cellular deformations, but not through large pores, was significantly impaired by colchicine. These data demonstrate that colchicine determines mechanics of neutrophils and, thereby, motility in confined spaces, which is crucial during extravasation of neutrophils in response to inflammatory stimuli. PMID:23901122

  2. Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

    International Nuclear Information System (INIS)

    We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for First time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype

  3. 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

  4. In situPCR on Plant Material with Sub-cellular Resolution

    DEFF Research Database (Denmark)

    Johansen, Bo

    1997-01-01

    In situPCR and reverse transcribedin situPCR have been tested on leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5% GA.In situPCR amplification of the gene coding for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was successfully performed following all three fixation protocols. As...... expected the PCR product was restricted to the plastids of all cells. Reverse transcribedin situPCR was performed onrbcL mRNA and in this case the PCR product was restricted to the plastids of the bundle sheath cells. This is the first report ofin situPCR on plant material and only the second report ofin...... situPCR with sub-cellular resolution.In situPCR onrbcL may prove to be a valuable positive control for futurein situPCR studies on plant material.Copyright 1997 Annals of Botany Company 10.1006/anbo.1997.0502...

  5. Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

    OpenAIRE

    Reddick, L. Evan; Alto, Neal M.

    2012-01-01

    The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our under...

  6. 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...

  7. Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator

    International Nuclear Information System (INIS)

    A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites

  8. Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

    OpenAIRE

    Wang, Yuanpeng; Huang, Jing; Gao, Yanzheng

    2012-01-01

    Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were g...

  9. 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...

  10. Multi-Scale Continuum Modeling of Biological Processes: From Molecular Electro-Diffusion to Sub-Cellular Signaling Transduction

    OpenAIRE

    Cheng, Y; Kekenes-Huskey, P; Hake, JE; Holst, MJ; McCammon, JA; Michailova, AP

    2012-01-01

    This article provides a brief review of multi-scale modeling at the molecular to cellular scale, with new results for heart muscle cells. A finite element-based simulation package (SMOL) was used to investigate the signaling transduction at molecular and sub-cellular scales (http://mccammon.ucsd.edu/smol/, http://FETK.org) by numerical solution of time-dependent Smoluchowski equations and a reaction-diffusion system. At the molecular scale, SMOL has yielded experimentally-validated estimates ...

  11. 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

  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. mGOASVM: Multi-label protein subcellular localization based on gene ontology and support vector machines

    OpenAIRE

    Wan Shibiao; Mak Man-Wai; Kung Sun-Yuan

    2012-01-01

    Abstract Background Although many computational methods have been developed to predict protein subcellular localization, most of the methods are limited to the prediction of single-location proteins. Multi-location proteins are either not considered or assumed not existing. However, proteins with multiple locations are particularly interesting because they may have special biological functions, which are essential to both basic research and drug discovery. Results This paper proposes an effic...

  14. 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/.

  15. 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.

  16. 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. PMID:26986088

  17. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    Science.gov (United States)

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi. PMID:20053634

  18. Influence of prey type on nickel and thallium assimilation, subcellular distribution and effects in juvenile fathead minnows (Pimephales promelas).

    Science.gov (United States)

    Lapointe, Dominique; Gentès, Sophie; Ponton, Dominic E; Hare, Landis; Couture, Patrice

    2009-11-15

    Because fish take up metals from prey, it is important to measure factors controlling metal transfer between these trophic levels so as to explain metal bioaccumulation and effects in fish. To achieve this, we exposed two types of invertebrates, an oligochaete (Tubifex tubifex) and a crustacean (Daphnia magna), to environmentally relevant concentrations of two important contaminants, nickel (Ni) and thallium (Tl), and fed these prey to juvenile fathead minnows (Pimephales promelas). We then measured the assimilation efficiency (AE), subcellular distribution and effects of these metals in fish. Fish assimilated dietary Tl more efficiently from D. magna than from T. tubifex, and more efficiently than Ni, regardless of prey type. However, the proportion of metal bound to prey subcellular fractions that are likely to be trophically available (TAM) had no significant influence on the efficiency with which fish assimilated Ni or Tl. In fish, the majority of their Ni and Tl was bound to subcellular fractions that are purportedly detoxified, and prey type had a significant influence on the proportion of detoxified Ni and Tl in fish. We measured higher activities of cytochrome C oxidase and glutathione S-transferase in fish fed D. magna compared to fish fed T. tubifex, regardless of the presence or absence of Ni or Tl in prey. However, we measured decreased activities of glutathione S-transferase and nucleoside diphosphate kinase in fish fed Tl-contaminated D. magna compared to fish from the three other treatment levels. PMID:20028068

  19. 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.)

  20. 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...

  1. 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

  2. Conversion of upland to paddy field specifically alters the community structure of archaeal ammonia oxidizers in an acid soil

    Directory of Open Access Journals (Sweden)

    M. S. Alam

    2013-08-01

    Full Text Available The function of ammonia-oxidizing archaea (AOA and bacteria (AOB depends on the major energy-generating compounds (i.e., ammonia and oxygen. The diversification of AOA and AOB communities along ecological gradients of substrate availability in a complex environment have been much debated but rarely tested. In this study, two ecosystems of maize and rice crops under different fertilization regimes were selected to investigate the community diversification of soil AOA and AOB upon conversion of an upland field to a paddy field and long-term field fertilization in an acid soil. Real-time quantitative polymerase chain reaction of ammonia monooxygenase (amoA genes demonstrated that the abundance of AOA was significantly stimulated after conversion of upland to paddy soils for more than 100 yr, whereas a slight decline in AOB numbers was observed. Denaturing gradient gel electrophoresis fingerprints of amoA genes further revealed remarkable changes in the community compositions of AOA after conversion of aerobic upland to flooded paddy field. Sequencing analysis revealed that upland soil was dominated by AOA within the soil group 1.1b lineage, whereas the marine group 1.1a-associated lineage predominated in AOA communities in paddy soils. Irrespective of whether the soil was upland or paddy soil, long-term field fertilization led to increased abundance of amoA genes in AOA and AOB compared with control treatments (no fertilization, whereas archaeal amoA gene abundances outnumbered their bacterial counterparts in all samples. Phylogenetic analyses of amoA genes showed that Nitrosospira cluster-3-like AOB dominated bacterial ammonia oxidizers in both paddy and upland soils, regardless of fertilization treatment. The results of this study suggest that the marine group 1.1a-associated AOA will be better adapted to the flooded paddy field than AOA ecotypes of the soil group 1.1b lineage, and indicate that long-term flooding is the dominant selective force

  3. Conversion of upland to paddy field specifically alters the community structure of archaeal ammonia oxidizers in an acid soil

    Science.gov (United States)

    Alam, M. S.; Ren, G. D.; Lu, L.; Zheng, Y.; Peng, X. H.; Jia, Z. J.

    2013-08-01

    The function of ammonia-oxidizing archaea (AOA) and bacteria (AOB) depends on the major energy-generating compounds (i.e., ammonia and oxygen). The diversification of AOA and AOB communities along ecological gradients of substrate availability in a complex environment have been much debated but rarely tested. In this study, two ecosystems of maize and rice crops under different fertilization regimes were selected to investigate the community diversification of soil AOA and AOB upon conversion of an upland field to a paddy field and long-term field fertilization in an acid soil. Real-time quantitative polymerase chain reaction of ammonia monooxygenase (amoA) genes demonstrated that the abundance of AOA was significantly stimulated after conversion of upland to paddy soils for more than 100 yr, whereas a slight decline in AOB numbers was observed. Denaturing gradient gel electrophoresis fingerprints of amoA genes further revealed remarkable changes in the community compositions of AOA after conversion of aerobic upland to flooded paddy field. Sequencing analysis revealed that upland soil was dominated by AOA within the soil group 1.1b lineage, whereas the marine group 1.1a-associated lineage predominated in AOA communities in paddy soils. Irrespective of whether the soil was upland or paddy soil, long-term field fertilization led to increased abundance of amoA genes in AOA and AOB compared with control treatments (no fertilization), whereas archaeal amoA gene abundances outnumbered their bacterial counterparts in all samples. Phylogenetic analyses of amoA genes showed that Nitrosospira cluster-3-like AOB dominated bacterial ammonia oxidizers in both paddy and upland soils, regardless of fertilization treatment. The results of this study suggest that the marine group 1.1a-associated AOA will be better adapted to the flooded paddy field than AOA ecotypes of the soil group 1.1b lineage, and indicate that long-term flooding is the dominant selective force driving the

  4. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  5. Solution structure of Pyrococcus furiosus RPP21, a component of the archaeal RNase P holoenzyme, and interactions with its RPP29 protein partner.

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2008-11-11

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5'-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentrations, four protein subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30, and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus ( Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha-helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step toward understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  6. 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 supplementation had no significant effects on the ruminal bacterial or archaeal community composition of finishing steers. PMID:27258373

  7. Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1

    International Nuclear Information System (INIS)

    A hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from S. solfataricus P1 has been crystallized as a recombinant protein with a vector-derived long N-terminal extension region. The P43212 crystals of recombinant ARF diffracted to 1.85 Å resolution using synchrotron radiation. The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule

  8. Abundance and Diversity of Bacterial, Archaeal, and Fungal Communities Along an Altitudinal Gradient in Alpine Forest Soils: What Are the Driving Factors?

    Science.gov (United States)

    Siles, José A; Margesin, Rosa

    2016-07-01

    Shifts in soil microbial communities over altitudinal gradients and the driving factors are poorly studied. Their elucidation is indispensable to gain a comprehensive understanding of the response of ecosystems to global climate change. Here, we investigated soil archaeal, bacterial, and fungal communities at four Alpine forest sites representing a climosequence, over an altitudinal gradient from 545 to 2000 m above sea level (asl), regarding abundance and diversity by using qPCR and Illumina sequencing, respectively. Archaeal community was dominated by Thaumarchaeota, and no significant shifts were detected in abundance or community composition with altitude. The relative bacterial abundance increased at higher altitudes, which was related to increasing levels of soil organic matter and nutrients with altitude. Shifts in bacterial richness and diversity as well as community structure (comprised basically of Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes) significantly correlated with several environmental and soil chemical factors, especially soil pH. The site at the lowest altitude harbored the highest bacterial richness and diversity, although richness/diversity community properties did not show a monotonic decrease along the gradient. The relative size of fungal community also increased with altitude and its composition comprised Ascomycota, Basidiomycota, and Zygomycota. Changes in fungal richness/diversity and community structure were mainly governed by pH and C/N, respectively. The variation of the predominant bacterial and fungal classes over the altitudinal gradient was the result of the environmental and soil chemical factors prevailing at each site. PMID:26961712

  9. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  10. Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

    Science.gov (United States)

    Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

    2016-09-01

    Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing. PMID:27338271

  11. 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

  12. Radioisotopic studies of the action of anticancer compounds in subcellular level

    International Nuclear Information System (INIS)

    Full text: Determination of mechanisms of biological action alkylating compounds with the purpose of an establishment of interrelation between structure and physiological action is one of the major problems of our time. Synthesis of new compounds and studying of corresponding processes in nucleus of cells is considered a priority of chemists and biologists. N-(β-chlorethyl)-salsoline has been earlier studied on antineoplastic activity in system in vitro, with the help marked thymidine. Thus it has been shown that this compound inhibits biosynthesis of nucleinic acids and proteins. In the present work influence of two antineoplastic preparations N-(β-chlorethyl)-salsoline and N-(β-chlorethyl)-hexamethylenimine on protein synthetic ability (PSA) at not sharing cells of animals of different age with use marked lysine has been investigated. Activity of these two preparations checked in identical concentration of 150 mkg/ml. Results of research have shown that N-(β-chlorethyl)-salsoline in a doze of 150 mkg/ml suppresses PSA nucleus of not sharing cells of a brain: on 11 % at 5-days animals; on 26 % at 25-days animals; on 14 % at 35 and 90-days animals. N-(β-chlorethyl)-hexamethylenimine in the same dozes on the contrary insignificantly stimulates this process. It is possible, that non-ribosomal alkylating does not occur in not sharing cells by these preparations (in normal cells). The action probably depends on structure of preparations. It is possible, that these preparations influence through immune system tumoral cells. Thus, antineoplastic compounds N-(β-chlorethyl)-salsoline and N-(β-chlorethyl)-hexamethylenimine do not operate on PSA at a subcellular level. Work is executed at financial support of the grant 'Research of natural connections with cancerolitic activity and creation on their basis of antineoplastic means'. This work was supported by the Center of Science and Technology RU (4F - 4.19)

  13. 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

  14. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    Science.gov (United States)

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis. PMID:26733305

  15. 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

  16. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    Directory of Open Access Journals (Sweden)

    Meredith J. Layton

    2014-04-01

    Full Text Available 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 maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v FBS. Following stimulation of RTKs (receptor tyrosine kinases, microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.

  17. Oxidative stress and the subcellular localization of the telomerase reverse transcriptase (TERT) in papillary thyroid cancer.

    Science.gov (United States)

    Muzza, Marina; Colombo, Carla; Cirello, Valentina; Perrino, Michela; Vicentini, Leonardo; Fugazzola, Laura

    2016-08-15

    During hormonogenesis, thyrocytes are physiologically exposed to high levels of oxidative stress (OS) which could either be involved in the pathogenesis of thyroid cancer or exert a cytotoxic effect. We analyzed the oxidative status of papillary thyroid cancer (PTC) both directly, by measuring H2O2 generation by NADPH oxidases (NOXs), and indirectly, by evaluating the antioxidant activity of glutathione peroxidase (GPX), which neutralizes H2O2 excess, and the lipid peroxidation (LP). Moreover, we investigated the subcellular localization of telomerase reverse transcriptase (TERT), and the H2O2 levels in the mitochondria of tumor and normal tissues. The calcium-dependent and independent H2O2 generation activity was significantly higher in tumors than in normal tissues. The GPX activity was higher in PTCs than in normal tissues, and, consistently, no differences were found in LP levels. Moreover, while TERT nuclear expression was similar in tumor and normal tissues, the mitochondrial localization was significantly higher in tumors. At the mitochondrial level, no differences were found in H2O2 generation between tumor and normal tissues. In conclusion, present data demonstrate that the intracellular H2O2 generation by NOXs is significantly higher in PTCs than in normal thyroid tissues. The increased GPX activity found in tumors counteracts the potential cytotoxic effects of high OS exposure. The significantly higher mitochondrial localization of TERT in tumors is consistent with its shuttling from the nucleus upon exposure to high OS. Finally, mitochondrial OS was not significantly different in tumors and normal tissues, supporting the postulated role of mitochondrial TERT in the control of local H2O2 production. PMID:27164443

  18. Identification of two subcellular sites for γ-glutamyltranspeptidase propeptide cleavage

    International Nuclear Information System (INIS)

    Renal intracellular and brush border membranes were purified from rats injected with [35S]methionine. Solubilized transpeptidase (γGT) was immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophresis (PAGE). The initial precipitates contain 3 bands corresponding to the core glycosylated propeptide (75K) and the mature heterodimer subunits (50K and 30K). The propeptide represents 75% of the radioactivity in γGT from 5 to 45 min postinjection consistent with cotranslational cleavage of 25% of the propeptide to subunits. By 20 min postinjection all three bands are more diffuse and endoglycosidase H-resistant. Between 20 and 30 min postinjection, propeptide and heterodimer coincidentally reach the brush border membrane. Propeptide then rapidly disappears (t/sub 1/2/ < 1 h) whereas heterodimer accumulates for 4 h then disappears with a t/sub 1/2/ of 2.5 d. The basis for these two distinct subcellular sites of γGT propeptide cleavage is unknown. Both purified γGT heterodimer and immunoprecipitates of labeled γGT occasionally exhibit high M/sub r/ bands (85K and 100K) on SDS-PAGE. Individual subunits and high M/sub r/ species were eluted from SDS gel slices, subjected to SDS-PAGE and analyzed on immunoblots with IgG affinity-purified against individual subunits. The cumulative data show that the subunits can form both stable homodimers (60K and 100K) and heterodimers (85K) during SDS-PAGE. Thus, these high M/sub r/ species do not represent biosynthetic intermediates of γGT

  19. Differential subcellular membrane recruitment of Src may specify its downstream signalling

    International Nuclear Information System (INIS)

    Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined

  20. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    Science.gov (United States)

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A. PMID:12514140