The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against ongogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cicle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed

The role of histidine-114 of Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase in chain-length determination

AbstractSulfolobus acidocaldarius geranylgeranyl diphosphate synthase yields (all-E)-C20 prenyl diphosphate as a final product. The three-dimensional model of the enzyme suggested that removing two bulky residues at 77 and 114 would allow additional prenyl-chain elongation. To test this, we examined several mutants with substitutions at 77 and/or 114. As a result, the mutants, F77G, F77G and H114A, F77G and H114G, H114A, and H114G gave C30, C45, C50, C30 and C40 as the main long product, respectively. These observations indicate that histidine-114 plays a crucial role in chain-length determination along with phenylalanine-77

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca^[2+]-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca^[2+]-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects

Hachisuka Mochiaki’s Overseas Achievements

Hachisuka Mochiaki (1846-1918) was the last lord of the Awa (Tokushima) domain, and he became a successful businessman and a statesman after the Meiji Restoration. He went to the UK to study when he was 25 years old, and stayed there for seven years, during which he graduated from college at the Balliol College, University of Oxford. Three years after returning to Japan, he was appointed to an envoy extraordinary and minister plenipotentiary of Japan in France (also serving as a minister in Spain, Portugal, Switzerland and Belgium) and he stayed in Paris three years. Despite ten years of foreign life, little has been known about his overseas activities. In this study, we investigated his overseas activities using digital archives of European libraries, and could find information from British Newspaper Archive, Welsh Newspaper Online, and Gallica. He began to attend public events in the UK after graduating from the Oxford University. He received in an audience by Queen Victoria and the Prince of Wales in 1877 and 1878, and attended to parties hosted by the Ministry of Foreign Affairs. He also participated in the launching ceremony of three Japanese warships Kongo, Fuso, and Hiei in 1877. He worked variously as a diplomat in Paris during 1884-1886. He signed three treaties, the Geneva Convention, the treaty on remittance by postal money order, and the International Meter Convention. Conpared with signing the Geneva Convention remaining two have not received much attention so far. In this study we found that he encouraged the Japanese government to join the International Meter Convention and negotiated with the Comité International des Poids et Mesures many times. He organized Japanese exhibition at a museum to introduce Japanese culture to Parisians, and he contributed to the academic exchange between France and Japan with the Geographical Society. And he and his wife Yoriko participated in many social events, and they held parties, concerts and theaters in Paris and Brussels. Several French newspapers mentioned elegant behavior of his wife Yoriko at those parties. And He focused on politics and business after returning to Japan, but also worked to promote exchange with foreign countries. In particular, he established the Welcome Society, which was the first organization in Japan for attracting and accommodating foreign tourists, with other of founders, and became the chairman of the organization. His abundant overseas experience and wide-ranging personal connections helped to establish this association

Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs

In most species, fertilization induces Ca(2+) transients in the egg. In mammals, the Ca(2+) rises are triggered by phospholipase Czeta (PLCzeta) released from the sperm; IP3 generated by PLCzeta induces Ca(2+) release from the intracellular Ca(2+) store through IP3 receptor, termed IP3-induced Ca(2+) release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 muM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca(2+) and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca(2+) sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca(2+) wave. [IP3] stayed at the elevated level with small peaks followed after Ca(2+) spikes through Ca(2+) oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca(2+) spike, suggesting that Ca(2+)-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca(2+) oscillations in fertilized eggs

Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of Ins P3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications. © 2015, Public Library of Science. All rights reserved

An overview of CRISPR-based tools and their improvements:new opportunities in understanding plant-pathogen interactions for better crop protection

Modern omics platforms have made the determination of susceptible/resistance genes feasible in any species generating huge numbers of potential targets for crop protection. However, the efforts to validate these targets have been hampered by the lack of a fast, precise and efficient gene targeting system in plants. Now, the repurposing of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has solved this problem. CRISPR/Cas9 is the latest synthetic endonuclease that has revolutionised basic research by allowing facile genome editing in prokaryotes and eukaryotes. Gene knockout is now feasible at an unprecedented efficiency with the possibility of multiplexing several targets and even genome-wide mutagenesis screening. In a short time, this powerful tool has been engineered for an array of applications beyond gene editing. Here we briefly describe the CRISPR/Cas9 system, its recent improvements and applications in gene manipulation and single DNA/RNA molecule analysis. We summarise a few recent tests targeting plant pathogens and discuss further potential applications in pest control and plant–pathogen interactions that will inform plant breeding for crop protection