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Characterization of E coli Hfq structure and its RNA binding properties

2005-12-07 , Sun, Xueguang

Hfq is a bacterial RNA-binding protein recently shown to contain the Sm motif, a characteristic of Sm proteins that function in RNA processing in archaea and eukaryotes. Hfq plays a major role in RNA-RNA interactions regulating translation. Comparative structural modeling and amino acid sequence alignment were used to predict the 3-D structure of Hfq and the model was in excellent agreement with the crystal structure which determined for S. aureus Hfq. The evolution of Hfq was explored by a BLAST search of microbial genomes followed by phyletic analysis. About half of the genomes examined contain at least one gene coding for Hfq. The presence and absence of Hfq closely followed major bacterial clades. The potential RNA binding residues on the two surfaces of the Hfq hexamer were proposed based on the bioinformatics studies and the mutant Hfq proteins with either single or double mutations on the two surfaces of the Hfq hexamer were generated. Their RNA binding properties was biophysically studied by gel-shift assay, fluorescence anisotropy and fluorescence quenching techniques. Results indicated that 1) point mutations on the distal surface of the Hfq hexamer, Y25A and K31A, have a major effect on A18 binding. Both reduce binding by about 1000 fold. Mutations on the proximal surface have a small or no influence on A18 binding. 2) Two mutations, F39A and R16A, on the proximal surface of the Hfq structure reduce binding to the DsrA domain II by 10 fold. Other mutations reduce binding by less than 2 fold. 3) An amino acid covariance was observed in L12 and F39. Mutation L12F can partially restore F39A in DsrA RNA binding. 4) It appears that two Hfq hexamers cooperatively bind one RNA for both DsrADII and A18.

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A characterization of the human G protein-coupled receptor, lysophosphatidic acid1 : its intracellular trafficking and signaling consequences on the tumor suppressor, P53

2005-04-26 , Murph, Mandi Michelle

Lysophosphatidic acid (LPA) is a mitogenic lipid that enhances cell growth, proliferation and motility through binding and activation of at least four receptors, LPA1/Edg2, LPA2/Edg4, LPA3/Edg7, and PPAR and #947;. Here, we show that LPA stimulation inhibits the cell cycle regulator and tumor suppressor, p53. Ten M LPA reduced the cellular levels of total p53 and p53 phosphorylated at serine 15 by approximately 50% in A549 cells and this effect was sustained for at least 6 h. This resulted in a corresponding decrease in p53-mediated transcription. Transient-transfection of the Edg-family LPA receptors, LPA1-3 in HepG2 cells, which do not respond to LPA, also showed this inhibitory response. The response was specific to LPA receptors since neither Gi-coupled M2 muscarinic acetylcholine receptors, nor a mutant LPA1 receptor (LPA1 R124A), which is unable to bind LPA, inhibited p53 activity. Both transient-transfection of the LPA-degrading lipid phosphate phosphatase-1 (LPP-1), or exogenous addition of phospholipase B, which decreases exogenous lysophosphatidate, reversed the LPA receptor-induced decrease in p53-mediated transcription. Although pertussis toxin did not prevent the inhibition of p53, a mutant LPA1 receptor (LPA1 and #8710;361), which lacks the C-terminal PDZ-binding domain, failed to inhibit p53 function. This establishes LPA-mediated inhibition of p53 function requires an interaction with PDZ-containing proteins. These data establish a novel role for LPA-mediated receptor activation in diminishing p53 activity; which, in addition to LPAs well-characterized effects on growth-promoting signaling pathways, is likely to contribute to the survival and proliferation of cancer cells. Of the Edg-family LPA receptors, the LPA1 receptor is the most widely expressed. In the next study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. LPA treatment induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removing agonist following incubation resulted in LPA1 recycling back to the surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). Finally, our results indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

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Plasticity of Consumer-prey Interactions in the Sea: Chemical Signaling, Consumer Learning, and Ecological Consequences

2004-11-23 , Long, Jeremy Dillon

Marine consumers and their prey display plasticity that affects the outcomes of their dynamic interactions as well as community structure and ecosystem function. Aquatic chemical signals induced plasticity in consumers and prey from a broad range of taxonomy (phytoplankton to fishes), sizes (microscopic to macroscopic), and habitats (pelagic to benthic), and this complex plasticity strongly affected consumer-prey interactions. Two fishes,

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Microbial diversity in sediments and gas hydrates associated with cold seeps in the Gulf of Mexico

2004-07-08 , Mills, Heath Jordan

A molecular phylogenetic approach was used to characterize the composition of microbial communities from two gas hydrate sedimentary systems in the Gulf of Mexico. Nucleic acids were extracted from three distinct locales on surface breaching gas hydrate mounds, i.e., sediment overlaying gas hydrate, sediment/hydrate interface and sediment-free hydrate, and from three sediment depths, i.e., 0-2, 6-8 and 10-12 cm, in Beggiatoa sp. mat-associated sediments located several meters from exposed gas hydrate. Samples were collected from a research submersible (water depth 550-575 m) during two research cruises aboard the R/V Seward Johnson I and II funded by the NSF Life in Extreme Environments program. The 16S rRNA gene and 16S rRNA were amplified using PCR and reverse transcription-PCR, respectively, from DNA and RNA extracted from the total microbial community. The primers targeted microorganisms at the domain-specific, i.e., Bacteria and Archaea, and group-specific, i.e., sulfate-reducing bacteria (SRB) and putative anaerobic methane-oxidizing (ANME) archaea, level. Sequence analysis of the Bacteria clones revealed that the microbial communities were primarily dominated by Deltaproteobacteria. Other Proteobacteria classes, including Epsilon- and Gammaproteobacteria, represented a large fraction of the total microbial community isolated from the sediment overlying hydrate sample and the metabolically active fraction of the 0-2 cm sediment depth sampled from the Beggiatoa sp. mat-associated sediments. Sequence analysis indicated the majority of the archaeal clones were most closely related to Methanosarcinales, Methanomicrobiales and distinct lineages within the ANME groups. Several novel lineages were identified including a fourth ANME-2 clade, i.e., ANME-2D, and three clades with no closely related previously sequenced 16S rRNA gene clones or isolates, i.e., Unclassified Bacteria groups 1 and 2 and Unclassified Euryarchaeota. These studies represent the first 16S rRNA gene and 16S rRNA phylogenetic-based description of microbial communities extant in sediment-free gas hydrate and in methane-rich hydrate-associated and Beggiatoa sp.-associated sediments from a hydrocarbon seep region in the Gulf of Mexico.

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Plant-herbivore interactions : consequences for the structure of freshwater communities and exotic plant invasions

2005-12 , Parker, John D.

Invasive exotic species threaten native biodiversity, alter ecosystem structure and function, and annually cost over $100 billion in the US alone. Determining the ecological traits and interactions that affect invasion success are thus critical for predicting, preventing, and mitigating the negative effects of biological invasions. Native herbivores are widely assumed to facilitate exotic plant invasions by preferentially consuming native plants and avoiding exotic plants. Here, I use freshwater plant communities scattered broadly across the Southeastern U.S. to show that herbivory is an important force driving the ecology and evolution of freshwater systems. However, native consumers often preferentially consume rather than avoid exotic over native plants. Analyses of 3 terrestrial datasets showed similar patterns, with native herbivores generally preferring exotic plants. Thus, exotic plants appear defensively nave against these evolutionarily novel consumers, and exotic plants may escape their coevolved, specialist herbivores only to be preferentially consumed by the native generalist herbivores in their new ranges. In further support of this hypothesis, a meta-analysis of 71 manipulative field studies including over 100 exotic plant species and 400 native plant species from terrestrial, aquatic, and marine systems revealed that native herbivores strongly suppressed exotic plants, while exotic herbivores enhanced the abundance and species richness of exotic plants by suppressing native plants. Both outcomes are consistent with the hypothesis that prey are susceptible to evolutionarily novel consumers. Thus, native herbivores provide biotic resistance to plant invasions, but the widespread replacement of native with exotic herbivores eliminates this ecosystem service, facilitates plant invasions, and triggers an invasional meltdown. Consequently, rather than thriving because they escape their co-evolved specialist herbivores, exotic plants may thrive because their co-evolved generalist herbivores have stronger negative effects on evolutionarily nave, native plants.

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Interactions between endogenous prions, chaperones and polyglutamine proteins in the yeast model

2005-03-16 , Gokhale, Kavita Chandan

Poly-Q expanded exon 1 of huntingtin (Q103) fused to GFP is toxic to yeast cells containing endogenous yeast prions, [PIN+] ([RNQ+]) and/or [PSI+], which presumably serve as aggregation nuclei. Propagation of yeast prions is modulated by the chaperones of Hsp100/70/40 complex. While some chaperones were reported to influence poly-Q aggregation in yeast, it was not clear whether they do it directly or via affecting yeast prions. Our data show that while dominant negative Hsp104 mutants antagonize poly-Q aggregation and toxicity by eliminating endogenous yeast prions, some mutant alleles of Hsp104 decreases size and ameliorate toxicity of poly-Q aggregates without affecting prion propagation. Elevated levels of the yeast Hsp40 proteins, Ydj1 and Sis1, exhibit opposite effects on poly-Q aggregation and toxicity without influencing prion propagation. Among the yeast Hsp70s, only overproduction of Ssa4 antagonized poly-Q toxicity. We have also isolated dominant Anti-poly-Q-toxicity (AQT) mutants counteracting poly-Q toxicity only in the absence of the major ubiquitin-conjugating enzyme Ubc4. Prion forming potential of other Q-rich proteins and influence of Q and P-rich regions on prion propagation were also studied. Our data connects poly-Q aggregation and toxicity to the stress defense pathway in yeast. As many stress-defense proteins are conserved between yeast and mammals, our data shed light on possible mechanisms modulating poly-Q aggregation and toxicity in mammalian cells.

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Regulation of Trichodesmium Nitrogen Fixation by Combined Nitrogen and Growth Rate: A Field and Culture Study

2004-11-21 , Holl, Carolyn Marie

Trichodesmium is a globally significant marine diazotroph responsible for supplying new nitrogen to the oligotrophic regions in which it is found. Though it has been studied for decades, our understanding of the ways in which environmental factors can affect its nitrogen fixation rate remains limited. A continuous culture of Trichodesmium was established in which steady state growth and nitrogen fixation were maintained at dilution rates ranging from 0.27 to 0.67 d-1. Our results clearly show that, as growth rate increased, biomass decreased linearly and nitrogen fixation rate increased linearly. C:N:P ratios remained constant over the range of growth rates studied, suggesting a tight coupling between macronutrient uptake and the maintenance of balanced growth at steady state. We used cultures at steady state to determine the impact of nitrate exposure and uptake on nitrogen fixation. Nitrate inhibits nitrogen fixation by up to 70% in a concentration-dependent manner at initial nitrate concentrations less than 10?? Nitrate uptake accounted for as much as 86% of total N uptake and, at initial nitrate concentrations greater than 2.5 ??more than made up for the observed inhibition of nitrogen fixation. A field study of this diazotroph shows that nitrogen fixation scales with light intensity from a maximum at 50% surface irradiance. Estimated areal nitrogen fixation rates in the Gulf of Mexico, based on vertical abundance profiles and the relationship between nitrogen fixation and surface irradiance, are comparable to measurements made in other oligotrophic regions. Stable isotopic composition of the particulate organic matter and the zooplankton confirms that Trichodesmium nitrogen and carbon are moving into the food chain and are important to higher trophic levels. As much as 60% of the zooplankton carbon was derived from Trichodesmium. Our work established that this diazotroph is ecologically important in the water column of the Gulf of Mexico, with important implications for nitrogen and carbon cycling. Findings from our field and culture studies can be added to models used to quantify the importance of Trichodesmium nitrogen fixation on an oceanic scale.

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Comparative Genomics of Microbial Signal Transduction

2005-11-28 , Ulrich, Luke

High-throughput genome processing, sophisticated protein sequence analysis, programming, and information management were used to achieve two major advances in the comparative genomics of microbial signal transduction. First, an integrated and flexible bioinformatics platform and the Microbial Signal Transduction database (MiST) were developed, which facilitated the genome-wide analysis of bacterial signal transduction. This platform was used successfully for the high-throughput identification and classification of signal transduction proteins in more than 300 archaeal and bacterial organisms. Second, analysis of information encoded in prokaryotic genomes revealed that the majority of signal transduction systems consist of one-component systems a single protein containing both input and output domains but lacking phosphotransfer domains typical of two-component systems. The prevalence of one-component systems is a paradigm-shifting discovery because two-component systems are currently viewed as the primary mode of signal transduction in prokaryotes. One-component systems are more widely distributed among bacteria and archaea and display a greater diversity of domains than two-component systems. Additionally, in-depth bioinformatic analyses were performed that further characterized the function of two, input, signaling domains. In summary, this systematic, high-throughput delineation of microbial signal transduction is another step forward in our understanding of the genomic basis of life.

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Characterization of DAP1/YPL170W: the Saccharomyces cerevisiae Membrane Associated Progesterone Receptor (MAPR)Homologue

2005-01-07 , Banna, Christopher David

Characterization of DAP1/YPL170W: the Saccharomyces cerevisiae Membrane Associated Progesterone Receptor (MAPR) Homologue Christopher D. Banna 135 pages Directed by Dr. Jung H. Choi MAPRs (Membrane Associated Progesterone Receptors) from several sources have been isolated, studied and minimally characterized in mammalian systems, yet the specific role of this protein family has not been fully determined. Early worked characterized this protein family as a type of steroid binding protein, unrelated to the classical nuclear receptors, and linked this family to non-genomic cellular responses. The MAPR homologues as a group have been suggested to play widely varying roles from axon guidance and neuronal formation, to steroid hydroxylation, to influencing reproductive behavior. Their specific role has not yet been clearly demonstrated in any organism. There is some debate as to whether MAPRs do indeed bind steroid compounds, but there is clear evidence this family of proteins is involved in steroid perception. Recent work has begun to link a specific member of the MAPR family, IZAg from rat, to steroid metabolism/production, specifically, in the hydroxylation step of glucocorticoid production from progesterone. In the yeast Saccharomyces cerevisiae, the MAPR homologue is DAP1. Preliminary work on haploid strains demonstrated several phenotypes associated with the DAP1 deletion mutant, most notably an altered sterol profile. Previous characterization of diploid homozygous mutant strain has shown a differential sensitivity to alcohol, an altered sterol profile, and a strong yeast two-hybrid interaction with Ypr118wp; methylthioribose-1-phosphate isomerase. Work in this study link the localization of Dap1p to lipid particles and on the ER, both sites of sterol synthesis. The sterol profiles of the control strain and the dap1Ġdeletion mutant strain were examined in detail. The most notable difference was the presence of an additional sterol compound associated with the deletion mutant strain. The structure of this compound does not correspond to normal sterols in the ergosterol biosynthetic pathway, but does correspond to structure of sterols in so-called alternate aberrant sterol pathways. The data presented in this study demonstrates that Dap1p was involved in sterol processing, although its specific role is unknown. Two possible scenarios are proposed; one where Dap1p is involved in regulating the flux of sterols from one internal membrane to another, and another where Dap1p is involved in aberrant sterol pathways.

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Biochemical and molecular characterization of streptococcus pneumoniae strains resistant to beta-lactam antibiotics

2004-07-09 , Korir, Cindy Chepngeno

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

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