Effects of nitrates and calcium channel blockers on Ca2+-ATPase in the microsomal fraction of porcine coronary artery smooth muscle cells

The effects of the antianginal drugs nitroglycerin, nicorandil, diltiazem, verapamil and nicardipine on the activity of calcium-stimulated magnesium-dependent ATPase (Ca2+-ATPase) were investigated in the microsomal fraction from porcine coronary artery smooth muscle cells. Two discrete Ca2+-dependent ATPase components were observed: [1] a high affinity component, which was a specific Ca2+-ATPase, [with a half saturation constant for Ca2+ (Km) of 0.44 microM, and maximum velocity (Vmax) of 124.3 pmol of phosphate (Pi) released/micrograms of protein/30 min]: [2] a low affinity component in which Ca2+ could be replaced by Mg2+ without loss of its activity. Nitroglycerin and nicorandil (1 microM and 10 microM) both stimulated the activity of the Ca2+-ATPase significantly [142 +/- 12 (mean +/- standard error), and 137 +/- 10% of the control with nitroglycerin, and 152 +/- 17 and 135 +/- 20% with nicorandil] at a Ca2+ concentration of 0.3 microM. Diltiazem, verapamil and nicardipine did not cause significant stimulation. Nitroglycerin and nicorandil (1 microM), significantly decreased the Km for Ca2+ from the control value of 0.44 +/- 0.06 microM to 0.26 +/- 0.03 and 0.22 +/- 0.03 microM, respectively. Nitroglycerin and nicorandil may dilate coronary arteries by stimulating this Ca2+ extrusion pump enzyme through reduction of intracellular Ca2+ in smooth muscle cells.     

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors.     

Purification and Properties of Polyol: NADP Oxidoreductase from Pichia quercuum

In order to explain the fermentation mechanism of xylitol production from d-xylose by Pichia quercuum, enzymatic study was carried out. Three kinds of enzymes that catalyzed the reduction of d-xylose to xylitol were purified from the extract of the yeast cells by ammonium sulfate fractionation, Sephadex G-200 gel filtration, hydroxylapatite chromatography and disc electrophoresis. The purification showed 27-fold, 135-fold and 93-fold increases of specific activities of reductase I, IIa and lib, respectively, over the crude extract. The reductase Ha was homogeneous in disc gel electrophoresis. The activity ratio of reductase I: IIa: IIb in the crude extract was estimated to be approximately 2: 1:1. The three enzymes were active with a variety of aldoses and had a specific requirement for NADPH. On the basis of the substrate specificity, coenzyme requirement and the stoichiometry of the reaction, the enzymes belong to polyol: NADP oxidoreductase (EC 1.1.1.21, trivial name, aldose reductase). The molecular weights for reductase I, IIa and IIb were estimated to be 160,000, 61,000 and 61,000, respectively, by gel filtration. Disc gel electrophoresis suggested that reductase Ila and lib were charge isomeric proteins with the same molecular size. Some other properties of the three enzymes were also described.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.