Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.     

Quantitative imaging using genetically encoded sensors for small molecules in plants

Quantitative imaging in live cells is a powerful method for monitoring the dynamics of biomolecules at an excellent spatio-temporal resolution. Such an approach, initially limited to a small number of substrates for which specific dyes were available, has become possible for a large number of biomolecules due to the development of genetically encoded, protein-based sensors. These sensors, which can be introduced into live cells through a transgenic approach, offer the benefits of quantitative imaging, with an extra advantage of non-invasiveness. In the past decade there has been a drastic expansion in the number of biomolecules for which genetically encoded sensors are available, and the functional properties of existing sensors are being improved at a dramatic pace. A number of technical improvements have now made the application of genetically encoded sensors in plants rather straightforward, and some of the sensors such as calcium indicator proteins have become standard analytical tools in many plant laboratories. The use of a handful of probes has already revealed an amazing specificity of cellular biomolecule dynamics in plants, which leads us to believe that there are many more discoveries to be made using genetically encoded sensors. In this short review, we will summarize the progress made in the past 15?years in the development in genetically encoded sensors, and highlight significant discoveries made in plant biology.     

Visualization of glutamine transporter activities in living cells using genetically encoded glutamine sensors

Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of F?ster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.     

Distribution of PERIOD-immunoreactive neurons and temporal change of the immunoreactivity under long-day and short-day conditions in the larval brain of the flesh fly Sarcophaga similis

The flesh fly Sarcophaga similis show a clear photoperiodic response; they develop into adults under long days, whereas they arrest their development at the pupal stage under short days. Although the involvement of a circadian clock in photoperiodic time measurement is suggested in this species, the anatomical location of the clock neurons responsible for the time measurement has been unknown. We detected two PERIOD-immunoreactive cell clusters in the larval brain; one cluster was located at the dorsoanterior region and the other at the medial region. We further investigated their temporal changes in PERIOD-immunoreactivity and compared their patterns under different photoperiods.     

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25°C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12–24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.     

Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed

A 7Crp peptide composed of seven major human T cell epitopes derived from the Japanese cedar pollen allergens Cry j 1 and Cry j 2 is an ideal tolerogen for peptide immunotherapy against Japanese cedar pollinosis. To maximize the accumulation level of the 7Crp peptide in transgenic rice seed, we tested endosperm specific promoters and intracellular localizations suitable for stable accumulation. A 7Crp peptide carrying the KDEL ER retention signal directed by the 2.3-kb promoter of the glutelin GluB-1, which contains a signal peptide, accumulated at the highest level of about 60 μg/grain. Notably, the 7Crp peptide predominantly accumulated in ER-derived protein bodies irrespective of the presence of various sorting signals or expression as a fusion protein with glutelin. We attribute this abnormal pattern of accumulation to the formation of disulfide bonds between the 7Crp peptide and cysteine-rich (Cys-rich) prolamin storage proteins. Furthermore, the formation of these aggregates induced the chaperone proteins BiP and PDI as an ER stress response.     

Analysis of ER stress in developing rice endosperm accumulating β‐amyloid peptide

The common neurodegenerative disorder known as Alzheimer’s disease is characterized by cerebral neuritic plaques of amyloid β (Aβ) peptide. Plaque formation is related to the highly aggregative property of this peptide, because it polymerizes to form insoluble plaques or fibrils causing neurotoxicity. Here, we expressed Aβ peptide as a new causing agent to endoplasmic reticulum (ER) stress to study ER stress occurred in plant. When the dimer of Aβ_1–42 peptide was expressed in maturing seed under the control of the 2.3‐kb glutelin GluB‐1 promoter containing its signal peptide, a maximum of about 8 μg peptide per grain accumulated and was deposited at the periphery of distorted ER‐derived PB‐I protein bodies. Synthesis of Aβ peptide in the ER lumen severely inhibited the synthesis and deposition of seed storage proteins, resulting in the generation of many small and abnormally appearing PB bodies. This ultrastructural change was accounted for by ER stress leading to the accumulation of aggregated Aβ peptide in the ER lumen and a coordinated increase in ER‐resident molecular chaperones such as BiPs and PDIs in Aβ‐expressing plants. Microarray analysis also confirmed that expression of several BiPs, PDIs and OsbZIP60 containing putative transmembrane domains was affected by the ER stress response. Aβ‐expressing transgenic rice kernels exhibited an opaque and shrunken phenotype. When grain phenotype and expression levels were compared among transgenic rice grains expressing several different recombinant peptides, such detrimental effects on grain phenotype were correlated with the expressed peptide causing ER stress rather than expression levels.     

Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26–41) alone is not enough, and that amino acids 12–41 from the N-terminus of ASB2a is essential for binding. ASB2a(12–41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12–41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.