Identification of lysophosphatidylthreonine with an aromatic fatty acid surrogate as a potent inducer of mast cell degranulation

Upon various stimulations, mast cells (MCs) release a wide variety of chemical mediators stored in their cytoplasmic granules, which then initiates subsequent allergic reactions. Lysophosphatidylserine (LysoPS), a kind of lysophospholipid, potentiates the histamine release from MCs triggered by antigen stimulation. We previously showed through structure-activity studies of LysoPS analogs that LysoPS with a methyl group at the carbon of the serine residue, i.e., lysophosphatidylthreonine (LysoPT), is extremely potent in stimulating the MC degranulation. In this study, as our continuing study to identify more potent LysoPS analogs, we developed LysoPS analogs with fatty acid surrogates. We found that the substitution of oleic acid to an aromatic fatty acid surrogate (C3-pH-p-O-C11) in 2-deoxy-1-LysoPS resulted in significant increase in the ability to induce MCs degranulation compared with 2-deoxy-1-LysoPS with oleic acid. Conversion of the serine residue into the threonine residue further increased the activity of MC degranulation both in vitro and in vivo. The resulting super agonist, 2-deoxy-LysoPT with C3-pH-p-O-C11, will be a useful tool to elucidate the mechanisms of stimulatory effect of LysoPS on MC degranulation.     

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.     

Characteristic seasonal variation in dissolved uranium concentration induced by the change of lake water pH in Lake Biwa,Japan

Epilimnion-dominated profiles of dissolved uranium (U) have been observed during summer in an oxygenated Japanese lake, Lake Biwa, contrary to the commonly accepted view that U shows conservative behavior in oxygenated seas and lakes. Monthly observations were conducted to reveal the mechanism for such characteristic distribution and geochemical behavior of dissolved U in the lake. In the surface water, dissolved U concentration started to increase in spring, peaked in summer, and decreased from autumn to winter. In contrast, the concentration remained almost constant in the middle layer (40 m depth) and decreased slightly in the bottom layer (70 m depth) throughout the stagnation period. Mass balance calculations of U suggest that the major mechanism for seasonal variations in the surface layer is the supply of U, not via water inflow from the watershed, but by internal chemical reactions within the lake. A laboratory experiment using the lake water and sediment demonstrated that U was desorbed from and adsorbed onto sediment in response to variations in lake water pH. From these results, it is inferred that the seasonal variation in the concentration of dissolved U in the epilimnion results mainly from the desorptive/adsorptive processes of U between sediment/water interface in response to variation in water pH, which is affected by biological activity in the lake.     

Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

Nondestructive evaluation of photosynthesis by delayed luminescence in Arabidopsis in Petri dishes

Nondestructive evaluation of photosynthesis is a valuable tool in the field and laboratory. Delayed luminescence (DL) can reflect charge recombination through the backflow of electrons. However, DL detection has not yet been adapted for whole plants in Petri dishes. To compensate for differences in DL decay between sibling Arabidopsis plants grown under the same conditions, we developed a time-sequential double measurement method. Using this method, we examined the influence of photosynthetic electron flow inhibitors, and differences in the DL decay curves were categorized by considering the initial and late phases of the decay curves, as well as their intermediate slopes. The appearance of concavity and convexity in DL curves in Arabidopsis was different from unicellular algae, suggesting complexity in the photosynthetic machinery of higher plants. This detection method should be invaluable for evaluating photosynthetic defects in higher plants under sterile conditions without interrupting plant culture.     

Environmental analysis of packaging‐derived changes in food production and consumer behavior

This study analyzed the environmental impacts of packaging‐derived changes in food production and consumer behavior to assist packaging designers in making environmentally conscious decisions. Packaging can be functionalized to prevent food loss and waste (FLW), for example, extending the expiration date and apportioning the package size, but it can generate additional environmental impacts from changes in food and packaging production. Previous studies assessed additional impacts from packaging production; however, the effects of packaging functionalization are yet to be connected with food production and consumer behavior. To examine the effect of functionalization on these aspects, we analyzed packaging‐derived changes in food production for milk and cabbage products. The case study compared products with functionalized packaging that permits a longer expiration date or a smaller portion size to their base‐case products. Our results showed that the packaging‐derived changes increased the global warming potential (GWP) of food production more than other processes did. Thus, changes in food production weakened the effectiveness of the packaging functionalization to decrease the GWP. Moreover, the analysis of consumer behavior scenarios showed that consumers’ perception of the expiration date decisively influences the effectiveness of packaging functionalization. When consumers discarded food after the expiration date, provided they consumed in small quantities, the packaging functionalization reduced FLW. From the scenario analysis, we identified appropriate combinations of packaging functionalization and consumer behaviors to effectively decrease total GWP. With our expanded analysis, packaging designers can understand the effectiveness of their decisions on the product life cycle in reducing FLW and environmental impacts.     

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche

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WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop

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