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#26860299   2016/02/10 Save this To Up

Solid State Structures of Alkali Metal Ion Complexes Formed by Low-Molecular-Weight Ligands of Biological Relevance.

This chapter provides structural data, mainly metal binding sites/modes, observed in crystal structures of alkali metal ion complexes containing low-molecular-weight ligands of biological relevance, mostly obtained from the Cambridge Structural Database (the CSD version 5.35 updated to February 2014). These ligands include (i) amino acids and small peptides, (ii) nucleic acid constituents (excluding quadruplexes and other oligonucleotides), (iii) simple carbohydrates, and (iv) naturally occurring antibiotic ionophores. For some representative complexes of these ligands, some details on the environment of the metal coordination and structural characteristics are described.

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#22210335   2012/01/02 Save this To Up

Nucleic acid-metal ion interactions in the solid state.

Metal ions play a key role in nucleic acid structure and activity. Elucidation of the rules that govern the binding of metal ions is therefore an essential step for better understanding of the nucleic acid functions. This review is as an update to a preceding one (Metal Ions Biol. Syst., 1996, 32, 91-134), in which we offered a general view of metal ion interactions with mono-, di-, tri-, and oligonucleotides in the solid state, based on their crystal structures reported before 1994. In this chapter, we survey all the crystal structures of metal ion complexes with nucleotides involving oligonucleotides reported after 1994 and we have tried to uncover new characteristic metal bonding patterns for mononucleotides and oligonucleotides with A-RNA and A/B/Z-DNA fragments that form duplexes. We do not cover quadruplexes, duplexes with metal-mediated base-pairs, tRNAs, rRNAs in ribosome, ribozymes, and nucleic acid-drug and -protein complexes. Factors that affect metal binding to mononucleotides and oligonucleotide duplexes are also dealt with.

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#20631160   2010/10/08 Save this To Up

Probabilistic analysis of gene expression measurements from heterogeneous tissues.

Tissue heterogeneity, arising from multiple cell types, is a major confounding factor in experiments that focus on studying cell types, e.g. their expression profiles, in isolation. Although sample heterogeneity can be addressed by manual microdissection, prior to conducting experiments, computational treatment on heterogeneous measurements have become a reliable alternative to perform this microdissection in silico. Favoring computation over manual purification has its advantages, such as time consumption, measuring responses of multiple cell types simultaneously, keeping samples intact of external perturbations and unaltered yield of molecular content.

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#19716528   2009/08/31 Save this To Up

In vivo circular RNA production using a constitutive promoter for high-level expression.

The permuted intron-exon (PIE) method based on group I intron self-splicing is the only methodology currently available for production of circular RNA in vivo. Here, we report improvement of the circular RNA expression method based on an induction-free vector system utilizing the highly efficient constitutive lpp promoter.

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#19305944   2009/05/08 Save this To Up

Thermovorax subterraneus, gen. nov., sp. nov., a thermophilic hydrogen-producing bacterium isolated from geothermally active underground mine.

A thermophilic, rod-shaped, motile, Gram-positive, spore-forming bacterium strain 70B(T) was isolated from a geothermally active underground mine in Japan. The temperature and pH range for growth was 50-81 degrees C (optimum 71 degrees C) and 6.2-9.8 (optimum pH 7-7.5), respectively. Growth occurred in the presence 0-2% NaCl (optimum 1% NaCl). Strain 70B(T) could utilize glucose, fructose, mannose, mannitol, pyruvate, cellobiose and tryptone as substrates. Thiosulfate was used as electron acceptor. Major whole-cell fatty acids were iso-C(15:0), C(16:0) DMA (dimethyl acetal), C(16:0) and anteiso-C(15:0). The G+C mol% of the DNA was 44.2%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the closest relatives of strain 70B(T) were Thermosediminibacter oceani DSM 16646(T) (94% similarity) and Thermosediminibacter litoriperuensis DSM 16647 (93% similarity). The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain 70B(T) represents a novel species in a new genus, for which the name Thermovorax subterraneus gen. nov., sp. nov. is proposed. The type strain of Thermovorax subterraneus is 70B(T) (=DSM 21563 = JCM 15541).

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#19208124   2009/02/11 Save this To Up

Comparison of Affymetrix data normalization methods using 6,926 experiments across five array generations.

Gene expression microarray technologies are widely used across most areas of biological and medical research. Comparing and integrating microarray data from different experiments would be very useful, but is currently very challenging due to the experimental and hybridization conditions, as well as data preprocessing and normalization methods. Furthermore, even in the case of the widely-used, industry-standard Affymetrix oligonucleotide microarrays, the various array generations have different probe sets representing different genes, hindering the data integration.

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#19138712   2009/03/02 Save this To Up

In vitro and in vivo production and purification of circular RNA aptamer.

RNA aptamers are potential candidates for RNA therapeutics. They must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. Since circular RNA molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin RNA aptamer both in vitro and in vivo. Circularization was accomplished by self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. This sequence was producible in both Escherichia coli cells and in vitro. The circularized streptavidin RNA aptamer retained its binding of streptavidin and was stabile in HeLa cell extracts compared to the linear form of the streptavidin aptamer. The self-spliced circular RNA from the transcribed permuted intron-exon transcripts in E. coli cells was purified from a total RNA fraction using the solid-phase DNA probe method following anion exchange chromatography that excluded gel electrophoresis. This study provides an alternative method for designing and purifying useful RNA aptamers.

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#18757075   2008/10/22 Save this To Up

Spatial and temporal changes in Actinobacterial dominance in experimental artificial groundwater recharge.

Artificial groundwater recharge (AGR) is used in the drinking water industry to supplement groundwater resources and to minimise the use of chemicals in water treatment. This study analysed the spatial and temporal changes of microbial communities in AGR using two test systems: a nutrient-amended fluidized-bed reactor (FBR) and a sand column. Structural changes in the feed lake water (Lake Roine), FBR, and sand column bacterial communities were determined by denaturing gradient gel electrophoresis (DGGE) and the length heterogeneity analysis of amplified 16S rRNA genes (LH-PCR). Two clone libraries were created to link the LH-PCR results to the dominant bacterial groups. The lake water bacterial community was relatively stable, with three bands dominating in all LH-PCR products. The most dominant fragment accounted for up to 72% and was derived from Actinobacteria. Based on the clone libraries and LH-PCR data, Actinobacteria also dominated in the unattached bacterial community of the FBR, whereas several Proteobacterial groups were more abundant on the FBR carrier particles. In the stabilised AGR system a major change in the community structure of the lake water bacteria took place during passage within the first 0.6m in the sand column as the community composition shifted from Actinobacteria-dominated populations to a diverse, mainly Proteobacterial communities. Concurrently, most of the dissolved organic carbon (DOC) was removed at this stage. In summary, the study showed that the make-up of microbial communities in experimental AGR systems responded to changes in their environment. LH-PCR showed potential as a method to determine microbial community dynamics in long-term studies at real-scale AGR sites. This is the first step to provide data on microbial community dynamics in AGR for drinking water production.

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#18442487   2008/06/20 Save this To Up

Age-related reductions in expression of serum response factor and myocardin-related transcription factor A in mouse skeletal muscles.

The molecular signaling pathways linking the atrophy of skeletal muscle during aging have not been identified. Using reverse transcription (RT)-PCR, Western blotting, and immunofluorescence microscopy, we investigated whether the amounts of RhoA, RhoGDI, SRF, MRTF-A, and MyoD in the triceps brachii and quadriceps muscles change with aging in mice. Young adult (3 mo) and aged (24 mo) C57BL/6J mice were used. Senescent mice possessed many fibers with central nuclei in the quadriceps muscle. Western blotting using a homogenate of whole muscle or the cytosolic fraction clearly showed that the amount of SRF protein was significantly decreased in the aged skeletal muscles. Immunofluorescence labeling indicated more SRF-positive muscle fibers in young mice. Both young and old mice possessed SRF immunoreactivity in some satellite cells expressing Pax7. MRTF-A and STARS mRNA levels significantly declined with aging in the triceps brachii and quadriceps muscles. The amount of MRTF-A protein was markedly reduced in the nuclear fraction of aged muscle of mice. The amounts of RhoA and RhoGDI in the crude homogenate or the cytosolic and membrane fractions were greater in the aged muscle. Senescent mice possessed significantly higher levels of MyoD protein in the cytosol and nucleus. Decreased SRF and MRTF expression may induce the atrophy of skeletal muscle with aging.

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#18029751   2007/11/21 Save this To Up

Production of circular streptavidin RNA aptamer in vivo.

RNA aptamers is one of highly hopeful candidates for RNA therapeutics. We previously reported an in vitro production of a circular streptavidin RNA aptamer. Here we show an application for producing the circular RNA aptamer in vivo. First, we constructed a circular RNA expression vector that contained self-splicing permuted intron-exon (PIE) sequences between T7 promoter and T7 terminator sequences so as to be transcribed by T7 RNA polymerase produced in JM109(DE3) cells. RNA expression driven by T7 RNA polymerase was triggered by addition of IPTG and the circularized RNA was generated from the resulting PIE transcripts. Circular streptavidin RNA aptamer generated in the JM109(DE3) cells was detected by a two dimensional denaturing PAGE analysis using the ethidium bromide staining. Northern blot analysis using a self-ligated sequence specific oligo DNA probe and sequencing analysis revealed that the self-splicing and circularization process precisely occurred in E. coli. The circular aptamer was easily purified by a solid phase DNA probe method from a partially purified total RNA fraction. This is the first demonstration of an in vivo expression of a circular RNA aptamer and its purification, paving the new way for inexpensive production of RNA aptamer.

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