Isolation and Maintenance.- Isolation and Differentiation of Medaka Embryonic Stem Cells.- Maintenance of Chicken Embryonic Stem Cells In Vitro.- Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro.- Derivation, Maintenance, and Characterization of Rat Embryonic Stem Cells In Vitro.- Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells.- Generation and Characterization of Monkey Embryonic Stem Cells.- Derivation and Propagation of Embryonic Stem Cells in Serum- and Feeder-Free Culture.- Signaling in Embryonic Stem Cell Differentiation.- Internal Standards in Differentiating Embryonic Stem Cells In Vitro.- Matrix Assembly, Cell Polarization, and Cell Survival.- Phosphoinositides, Inositol Phosphates, and Phospholipase C in Embryonic Stem Cells.- Cripto Signaling in Differentiating Embryonic Stem Cells.- The Use of Embryonic Stem Cells to Study Hedgehog Signaling.- Transfection and Promoter Analysis in Embryonic Stem Cells.- SAGE Analysis to Identify Embryonic Stem Cell-Predominant Transcripts.- Utilization of Digital Differential Display to Identify Novel Targets of Oct3/4.- Gene Silencing Using RNA Interference in Embryonic Stem Cells.- Genetic Manipulation of Embryonic Stem Cells.- Efficient Transfer of HSV-1 Amplicon Vectors Into Embryonic Stem Cells and Their Derivatives.- Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells.- Use of the Cytomegalovirus Promoter for Transient and Stable Transgene Expression in Mouse Embryonic Stem Cells.- Use of Simian Immunodeficiency Virus Vectors for Simian Embryonic Stem Cells.- Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells.- DNA Damage Response and Mutagenesis in Mouse Embryonic Stem Cells.- Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro.- Use of Embryonic Stem Cells in Pharmacological and Toxicological Screens.- Use of Differentiating Embryonic Stem Cells in Pharmacological Studies.- Embryonic Stem Cells as a Source of Differentiated Neural Cells for Pharmacological Screens.- Use of Murine Embryonic Stem Cells in Embryotoxicity Assays.- Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells.- Epigenetic Analysis of Embryonic Stem Cells.- Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion.- Methylation in Embryonic Stem Cells In Vitro.- Tumor-Like Properties.- Identification of Genes Involved in Tumor-Like Properties of Embryonic Stem Cells.- In Vivo Tumor Formation From Primate Embryonic Stem Cells.- Animal Models and Therapy.- Directed Differentiation and Characterization of Genetically Modified Embryonic Stem Cells for Therapy.- Use of Differentiating Embryonic Stem Cells in the Parkinsonian Mouse Model.

Isolation and characterization

Halophytes, plants that survive to reproduce in environments where the salt concentration is around 200 mM NaCl or more, constitute about 1% of the worlds flora. Some halophytes show optimal growth in saline conditions; others grow optimally in the absence of salt. However, the tolerance of all halophytes to salinity relies on controlled uptake and compartmentalization of Na+, K+ and Cl- and the synthesis of organic compatible solutes, even where salt glands are operative. Although there is evidence that different species may utilize different transporters in their accumulation of Na+, in general little is known of the proteins and regulatory networks involved. Consequently, it is not yet possible to assign molecular mechanisms to apparent differences in rates of Na+ and Cl- uptake, in root-to-shoot transport (xylem loading and retrieval), or in net selectivity for K+ over Na+. At the cellular level, H+-ATPases in the plasma membrane and tonoplast, as well as the tonoplast H+-PPiase, provide the transmembrane proton motive force used by various secondary transporters. The widespread occurrence, taxonomically, of halophytes and the general paucity of information on the molecular regulation of tolerance mechanisms persuade us that research should be concentrated on a number of model species that are representative of the various mechanisms that might be involved in tolerance.

Salinity tolerance in halophytes

Developments in industrially important thermostable enzymes: a review.

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Iron-chelating therapy and the treatment of thalassemia

The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.

Activation of AtMEK1, an Arabidopsis mitogen‐activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: mixed exposure to phenol, phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei.

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

Abscisic acid (ABA) is a phytohormone that regulates many physiological functions, such as plant growth, development and stress responses. The MAPK cascade plays an important role in ABA signal transduction. Several MAPK and MAPKK molecules are reported to function in ABA signaling; however, there have been few studies related to the identification of MAPKKK upstream of MAPKK in ABA signaling. In this study, we show that an Arabidopsis MAPKKK, MAPKKK18 functions in ABA signaling. The expression of MAPKKK18 was induced by ABA treatment. Yeast two-hybrid analysis revealed that MAPKKKK18 interacted with MKK3, which interacted with C-group MAPK, MPK1/2/7. Immunoprecipitated kinase assay showed that the 3xFlag-tagged MAPKKK18, expressed in Arabidopsis plants, was activated when treated with ABA. These results indicate the possibility that the MAPK cascade is composed of MAPKKK18, MKK3 and MPK1/2/7 in ABA signaling. The transgenic plants overexpressing MAPKKK18 (35S:MAPKKK18) and its kinase negative mutant (35S:MAPKKK18 KN) were generated, and their growth was monitored. Compared with the WT plant, 35S:MAPKKK18 and 35S:MAPKKK18 KN showed smaller and bigger phenotypes, respectively. Senescence of the rosette leaves was promoted in 35S:MAPKKK18, but suppressed in 35S:MAPKKK18 KN. Furthermore, ABA-induced leaf senescence was accelerated in 35S:MAPKKK18. These results suggest that MAPKKK18 controls the plant growth by adjusting the timing of senescence via its protein kinase activity in ABA dependent manners.

An abscisic acid inducible Arabidopsis MAPKKK, MAPKKK18 regulates leaf senescence via its kinase activity

The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca2+ signaling occurred upstream of the MEKK1–MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.

Takashi Nanmori

Papers

Activation of AtMEK1, an Arabidopsis mitogen‐activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: mixed exposure to phenol, phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei.

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

An abscisic acid inducible Arabidopsis MAPKKK, MAPKKK18 regulates leaf senescence via its kinase activity

Phosphorylation of Arabidopsis thaliana MEKK1 via Ca 2+ signaling as a part of the cold stress response