Complete genome sequence of NBRC 3288, a unique cellulose-nonproducing strain of Gluconacetobacter xylinus isolated from vinegar

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.     

A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca²⁺. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca²⁺ than with it. Ca²⁺-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.     

Interrupted 2'-o,4'-C-aminomethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'- BNA(NC)) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2',4'-BNA(NC) modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2',4'-BNA(NC) modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2',4'-BNA(NC) modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2',4'-BNA(NC)-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2',4'-BNA(NC)-modified positions in TFO. We conclude that the interrupted 2',4'-BNA(NC) modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.     

High-throughput screening for small-molecule adiponectin secretion modulators

Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor γ (PPARγ) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPARγ agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes for discovery of adiponectin secretion modulators. They screened a library of approximately 100 000 small-molecule compounds using this model, performed several follow-up screens, and identified six hit compounds that increase adiponectin secretion without having PPARγ agonistic activity. These compounds may be useful drug candidates for diabetes, obesity, atherosclerosis, and other metabolic syndromes. This HTS assay might be applicable to screening for other adipokine modulators that can be useful for the treatment of other conditions.     

In situ expression of the mitochondrial ATPase6 gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.     

Modifying the LEACHM model for process-based prediction of nitrate leaching from cropped Andosols

Aims

Nitrogen (N) management strategies for reducing the risk of groundwater contamination around agricultural fields require precise prediction of N leaching using a process-based model. We modified LEACHM model for use in Andosols, which are characterized by slow soil organic carbon (SOC) mineralization and nitrate adsorption.

Methods

The modification was made with regard to the SOC mineralization of incoming plant-residue/manure and humus following the RothC model, as well as for nitrate adsorption. Empirical equations were employed to determine the parameters of the modified model. The ability of the modified LEACHM to predict N leaching was tested against existing data from a 4-year lysimeter study for cropped Andosol and sandy soils and compared with that of the original model.

Results

The modified model improved the prediction of leached N concentrations and the loss of N from Andosol with relative improvements of 63.5 and 76.5 %, respectively, over the original model, while retaining model applicability in sandy soil. This effective modeling was achieved by using precise predictions of N mineralization in the humus pool along with SOC mineralization processes that were based on the RothC model.

Conclusions

The modification extended the applicability of LEACHM and may provide better N management strategies for reducing leaching from cultivated Andosols.     

Chiral HPLC Separation,Absolute Structural Elucidation,and Determination of Stereochemical Stability of trans‐Bis[2‐(2‐pyridinyl)aminophenolato] Cyclotriphosphazene

Chiral high‐performance liquid chromatography (HPLC) separation of trans‐bis[2‐(2‐pyridyl)aminophenolato] dichlorocyclotriphosphazene 1 was achieved and the absolute configuration of (+)-1 was assigned to be S,S by single‐crystal X‐ray structural analysis. The optically pure 1,2‐diphenyl‐1,2‐ethanediolate derivatives (+)‐ 2a and (?)‐ 2b were synthesized by the reactions of (+)-1 and (-)-1 with (R,R)‐hydrobenzoin, respectively, in refluxing toluene in the presence of an excess amount of triethylamine and a catalytic amount of 4‐(dimethylamino)pyridine. The racemization of the enantiomers of 1 and the epimerization of diastereomers of 2 were not observed in refluxing toluene neither under acidic nor basic conditions. The stereochemistry of (+)-1 was confirmed by the crystal structure of (+)‐ 2a and bis[(4‐methyl‐2‐pyridyl)oxy]cyclotriphosphazene (+)-3 derived from (+)-1 . Chirality 28:556–561, 2016. © 2016 Wiley Periodicals, Inc.     

Phosphorylated ubiquitin chain is the genuine Parkin receptor

PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria.