Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca²⁺-dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE-cellulose, Sephadex G-200, and calmodulin-affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a K _a value of 16.7 n m . The K _a values of peaks I and II for Ca²⁺ were 2.4 and 1.6 μ m , respectively.     

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.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

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.     

Evaluation of high sensitive C-reactive protein and 5-10 methylenetetrahydrofolate reductase genotype in Japanese young adults

Primary objective: To carry out a preliminary evaluation of subclinical inflammation and its genetic background in young adults.

Research design: Fifty-five healthy Japanese young adults aged 19–27 years (37 males and 18 females, mean age: 22.3 years), and 58 healthy Japanese adults aged 40 to 60 years (21 males and 37 females, mean age: 51.5 years) were included in this study.

Methods and procedures: We measured plasma high-sensitive C-reactive protein (hs-CRP) levels and screened for the C677T polymorphism of the 5-10 methylenetetrahydrofolate reductase gene (MTHFR), which is considered a genetic risk factor for atherosclerosis, by HinfI digestion.

Main outcomes and results: Hs-CRP levels of the young adult group were significantly lower than the levels of the middle aged group (0.014±0.030 mg/dl vs. 0.031±0.040 mg/dl, p=0.005). The levels were significantly higher in males than in females (0.028±0.019 mg/dl vs. 0.013±0.010 mg/dl, p=0.008) among young adults. Furthermore, we evaluated the relationship of the C677T genotype and hs-CRP values, but found no association between them.

Conclusions: Although the sample size is limited, our preliminary study demonstrated the profiles of hs-CRP in Japanese young adults. Further investigation will be needed to establish the guidelines for customized school health education using sensitive laboratory and genetic markers.     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.