β-Catenin-Driven Binary Fate Specification Segregates Germ Layers in Ascidian Embryos

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Highlights? β-catenin nuclear asymmetry after animal-vegetal-oriented cell divisions ? β-catenin nuclear asymmetry drives binary cell fate choices ? Combinatorial codes of nuclear β-catenin activation segregate ascidian germ layers ? Nuclear β-catenin ON-to-OFF activity is required for marginal mesoderm formation     

Cloning of the Genes Concerned in Phenylalanine Biosynthesis in Corynebacterium glutamicum and its Application to Breeding of a Phenylalanine Producing Strain

The chorismate mutase and prephenate dehydratase genes of phenylalanine producing Corynebacterium glutamicum K38, which is resistant to p-fluorophenylalanine and m-fluorophenylalanine, were cloned into plasmid pCE53 in C. glutamicum KY9456, which lacks chorismate mutase and prephenate dehydratase. One of the resultant plasmids, pCmB4, contained a 9.4kb BamHI DNA fragment inserted into the unique BamHl site of pCE53. Plasmid pCmB4 complemented a phenylalanine and tyrosine double auxotroph of C. glutamicum KY9456. Introduction of pCmB4 into C. glutamicum RRL5 resulted in an about ten times increase in chorismate mutase activity. C. glutamicum K38 carrying the plasmid accumulated 19.0mg/ml of phenylalanine (50% increase over the yield of K38).     

Essential Role of Aspartokinase in L-Threonine Production by Escherichia coli W Mutants

We previously constructed an l-threonine-producing strain of E. coli W, KY8280, which is an Ile⁺ revertant of KY8279 which requires l-methionine, a,£-diaminopimelic acid and l-isoleucine [H. Kase et al., Agric. Biol. Chem., 35, 2089 (1971)]. From KY8280, another l-threonine-hyperproducing strain, KY8366, was obtained as an α-amino-β-hydroxy valeric acid (AHV, a threonine analog)-resistant mutant. Enzymatic analysis revealed that KY8280 constitutively expressed 8-fold higher l-threonine-sensitive aspartokinase I activity than KY8279. In addition, KY8366 constitutively expressed 13-fold higher l-lysine-sensitive aspartokinase III activity than KY8280. Such elevated levels of aspartokinases may contribute to the hyperproduction of l-threonine by these mutant strains. KY8366 produced 28 mg/ml of l-threonine in a culture medium fed with 12% glucose.     

Phosphorylation of Arabidopsis thaliana MEKK1 via Ca2+ signaling as a part of the cold stress response

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.     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

TcSCA complements yeast mutants defective in Ca2+ pumps and encodes a Ca2+-ATPase that localizes to the endoplasmic reticulum of Trypanosoma cruzi

Intracellular Ca(2+) in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca(2+)-ATPase. Evidence for an endoplasmic reticulum-located Ca(2+) uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca(2+)-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca(2+) pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca(2+)-dependent and hydroxylamine-sensitive (32)P-labeled phosphoprotein of 110 kDa in the presence of [gamma-(32)P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn(2+) suggests that TcSCA may also regulate Mn(2+) homeostasis by pumping Mn(2+) into the endoplasmic reticulum of T. cruzi.