Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Antibody against neurofibromatosis type 1 gene product reacts with a triton-insoluble GTPase activating protein toward ras p21

Cellular fractionation of GTPase activating protein (GAP) activity using bovine cerebral cortex revealed that about half of GAP activity was found in membrane fraction. GAP activity of membrane was not solubilized with 0.5% (v/v) triton X-100 and was immunoprecipitated with antibody against carboxy-terminus of neurofibromatosis type 1 (NF1) gene product. In contrast, soluble GAP activity was precipitated with antibody against GAP but not with anti-NF1. These results suggest that NF1 gene product is a GTPase activating protein toward ras p21 with completely different intracellular distribution from that of GAP.     

Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.     

Impact of Noncovalent Sulfur–Fluorine Interaction Position on Properties,Structures, and Photovoltaic Performance in Naphthobisthiadiazole‐Based Semiconducting Polymers

Controlling the energetics and backbone order of semiconducting polymers is essential for the performance improvement of polymer‐based solar cells. The use of fluorine as the substituent for the backbone is known to effectively deepen the molecular orbital energy levels and coplanarize the backbone by noncovalent interactions with sulfur of the thiophene ring. In this work, novel semiconducting polymers are designed and synthesized based on difluoronaphthobisthiadiazole (FNTz) as a new family of naphthobisthiadiazole (NTz)–quaterthiophene copolymer systems, which are one of the highest performing polymers in solar cells. The effect of the fluorination position on the energetics and backbone order is systematically studied. It is found that the dependence of the solar cell fill factor on the active layer thickness is very sensitive to the fluorination position. It is thus further investigated and discussed how the structural features of the polymers influence the photovoltaic parameters as well as the diode characteristics and bimolecular recombination. Further, the polymer with fluorine on both the naphthobisthiadiazole and quaterthiophene moieties exhibits a quite high power conversion efficiency of 10.8% in solar cells in combination with a fullerene. It is believed that the results would offer new insights into the development of semiconducting polymers.     

Purification Process for the Preparation and Characterizations of Hen Egg White Ovalbumin,Lysozyme, Ovotransferrin,and Ovomucoid

Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.     

Monte Carlo simulation and measurement of radiation leakage from applicators used in external electron radiotherapy

External electron radiotherapy is performed using a cone or applicator to collimate the beam. However, because of a trade-off between collimation and scattering/bremsstrahlung X-ray production, applicators generate a small amount of secondary radiation (leakage). We investigate the peripheral dose outside the radiation field of a Varian-type applicator. The dose and fluence outside the radiation field were analyzed in a detailed Monte Carlo simulation. The differences between the calculation results and data measured in a water phantom in an ionization chamber were less than ±1% in regions more than 3 mm below the surface of the phantom and at the depth of dose maximum. The calculated fluence was analyzed inside and outside the radiation field on a plane just above the water phantom surface. Changing the electron energy affected the off-axis fluence distribution outside the radiation field; however, the size of the applicator had little effect on this distribution. For each energy, the distributions outside the radiation field were similar to the dose distribution at shallow depths in the water phantom. The effect of secondary electrons generation by photon transmission through the alloy making up the lowest scraper was largest in the region from the field edge to directly below the cutout and at higher beam energies. The results of the Monte Carlo simulation confirm that the peripheral dose outside the field is significantly affected by radiation scattered or transmitted from the applicator, and the effect increases with the electron energy.     

Synthesis of an Oligonucleotide Bearing a Phosphate Function at the 5′-Terminus Using a Novel Protecting Group in Terms of a Cellulose Acetate Derivative as a Polymer-Support

Abstract

Synthesis of the title oligonucleotide bearing a phosphate function at the 5′-terminus, i.e., pCpUpCpGpUpCpCpApCpCpA, by the use of the terminal cytidylic acid unit involving a novel protecting group of 2-[2-(monomethoxytrityloxy)ethylthio]ethyl group on its 5′-phosphoryl function in terms of a cellulose acetate polymer-support is described.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.