Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.     

The roles of circRFWD2 and circINO80 during NELL‐1‐induced osteogenesis

Bone defects caused heavy social and economic burdens worldwide. Nel‐like molecule, type 1 (NELL‐1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL‐1‐induced osteogenesis of human adipose‐derived stem cells (hASCs). In this study, we performed RNA‐sequencing to investigate the expression profiles of circRNAs in recombinant NELL‐1‐induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up‐regulated during recombinant NELL‐1‐induced osteogenesis, and knockdown of them affected the positive effect of NELL‐1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa‐miR‐6817‐5p, which could inhibit the osteogenesis. Silencing hsa‐miR‐6817‐5p could partially reverse the negative effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL‐1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.     

Calculations of the C<Subscript>2</Subscript> fragmentation energies of higher fullerenes C<Subscript>80</Subscript> and C<Subscript>82</Subscript>

The C₂ fragmentation energies of the most stable isolated-pentagon-rule (IPR) isomers of the C₈₀ and C₈₂ fullerenes were evaluated with second-order Møller-Plesset (MP2) theory, density-functional theory (DFT) and the semiempirical self-consistent charge density-functional tight-binding (SCC-DFTB) method. Zero-point energy, ionization energy and empirical C₂ corrections were included in the calculation of fragmentation energies for comparison with experimental C₂ fragmentation energies of the fullerene cations. In the case of the most probable Stone-Wales pathway of C₂ fragmentation of C₈₀, the calculated \(D_{0} {\left( {{\text{C}}_{{{\text{80}}}} ^{ + } } \right)}\) agree well with experimental data, whereas in the case of C₈₂ fragmentation, the calculated \(D_{0} {\left( {{\text{C}}_{{{\text{82}}}} ^{ + } } \right)}\) exceed by up to 1.2 eV the experimental ones, which suggests that other IPR isomers may be present in sufficient amounts in experimental samples. Computer-intensive MP2 calculations and DFT calculations with larger basis sets do not yield much improved C₂ fragmentation energies, compared to those reported earlier with B3LYP/3-21G. On the other hand, semiempirical approaches such as SCC-DFTB, which are orders of magnitude less intensive, yield satisfactory fragmentation energies for higher fullerenes and may become a method of choice for routine calculations of fullerenes and carbon nanotubes.

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Figure C₂ fragmentation energies of C₈₀ and C₈₂ fullerenes have been calculated with B3LYP/6-31G* model chemistry, with semiempirical self-consistent-charge density-functional tight-binding (SCC-DFTB) method and with the more rigorous MP2 method. The influence of basis set extension and level of theory on the resulting fragmentation energies is discussed

     

In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins

Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family. Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily. We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily. Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily. This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function. The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution.     

Ku80 facilitates chromatin binding of the telomere binding protein,TRF2

The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome, and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.     

Ku80 facilitates chromatin binding of the telomere binding protein,TRF2

The Ku70/80 heterodimer is central to non-homologous end joining repair of DNA double-strand breaks and the Ku80 gene appears to be essential for human but not rodent cell survival. The Ku70/80 heterodimer is located at telomeres but its precise function in telomere maintenance is not known. In order to examine the role of Ku80 beyond DNA repair in more detail, we have taken a knockdown approach using a human fibroblast strain. Following targeted Ku80 knockdown, telomere defects are observed and the steady state levels of the TRF2 protein are reduced. Inhibitor studies indicate that this loss of TRF2 is mediated by the proteasome and degradation of TRF2 following Ku depletion appears to involve a decrease in chromatin binding of TRF2, suggesting that the Ku heterodimer enhances TRF2 chromatin association and that non-chromatin bound TRF2 is targeted to the proteasome.Key words: Ku80, TRF2, chromatin, telomere, fibroblast     

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.     

GPR80/99, proposed to be the P2Y<Subscript>15</Subscript> receptor activated by adenosine and AMP,is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y₁₅ receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca²⁺ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9). However, the cell line (HEK293) used to carry out those studies endogenously expresses A_2A and A_2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, -ketoglutarate. Consistent with this report, -ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts -ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by -ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.     

Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy

In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-Å cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the γ-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.     

Deletion of the phage lambda att80 Tn9 genome; the type of intramolecular recombination

The intramolecular deletion-generating recombination which transforms lambda bacteriophage genomes into the plasmids (named pLS) proved to be site-specific to a certain extent. Using electron microscopy heteroduplex analysis three preferential sites for this recombination were found in seven independent pLS isolates studied. Att-sites were not registered to be involved in the formation of deletions in isolates studied. It was shown that recombination operating in our system was independent of the phage int and bacterial recA genes.     

Haemoglobin F Victoria Jubilee (alpha 2 A gamma 2 80 Asp-Try).

A new A gamma chain haemoglobin variant, haemoglobin F Victoria Jubilee, with an electrophoretic mobility slightly anodal to haemoglobin F Port Royal, was found in a Jamaican infant. The amino acid residue substitution of 80 Aspartic Acid leads to Tyrosine was associated with alanine in position 136. Haemoglobin F Victoria Jubilee constituted about 7.0 percent of the total haemoglobin F.     

Adsorption Behavior of Tween 80 on Soil in the Presence of CdCl2 and DDT

Batch experiments were conducted to investigate the adsorption behavior of Tween 80 in the systems composed of Tween 80, CdCl₂, and/or DDT. The results show that Cd²⁺ from CdCl₂ is the functional fraction influencing the adsorption of Tween 80 to soil, rather than Cl^?. Moreover, DDT can induce the increase of the critical micelle concentration (CMC) of Tween 80, which further impacts the Tween 80 adsorption behavior. The Tween 80 adsorption to soil in the Cd²⁺-DDT coexisted system follows the Langmuir isotherm, as in the Tween 80-Cd²⁺ or -DDT systems. Cd²⁺ and/or DDT decrease(s) the adsorption capacity of Tween 80 to soil, and the magnitude of decrease is dependent on the concentration of coexisting pollutants. Although DDT has a stronger inhibitory effect on Tween 80 adsorption than Cd²⁺ under the same DDT/Cd²⁺ concentrations, the coexistence of Cd²⁺ and DDT has an antagonistic effect on the adsorption of Tween 80. This effect is impacted by the concentrations of the coexisting pollutants, and is a result of the complex interaction among the three pollutants.     

Conformational Correlates of the Epitopes of Human Myelin Basic Protein Peptide 80–89

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.