Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, alpha-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.     

Expression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes,mice, and humans

Receptor tyrosine kinases (RTKs) play crucial roles in various developmental processes. Ror-family RTKs are characterized by the intracellular tyrosine kinase domains, highly related to those of the Trk-family RTKs, and by the extracellular Frizzled-like cysteine-rich domains (CRDs) and Kringle domains. Rors are evolutionally conserved among Caenorhabditis elegans, Aplysia, Drosophila melanogaster, Xenopus, mice, and humans. In D. melanogaster and mammals, pairs of structurally related Rors are found, while a single Ror protein is identified in C. elegans or Aplysia. In Aplysia and D. melanogaster, Rors are expressed exclusively in developing nervous systems. On the other hand, rather widespread expression of Rors was observed in C. elegans and mammals. Mutations in Ror of C. elegans cause inappropriate axon outgrowth as well as defects in cell migration and asymmetric cell division. It has also been reported that the nematode Ror possesses kinase-dependent and kinase-independent functions. Mouse Rors, Ror1, and Ror2, are expressed mainly in migrating neural crest cells and mesenchymal cells, and Ror2-deficient mice exhibit skeletal abnormalities and ventricular septal defects in the heart. Although Ror1-deficient mice exhibit no apparent skeletal or cardiac abnormalities, Ror1/Ror2 double mutant mice show markedly enhanced skeletal and cardiac abnormalities compared with Ror2 mutant mice, indicating genetic interaction of Ror1 and Ror2. In humans, mutations within Ror2 have been found in two genetic skeletal disorders, recessive Robinow syndrome and dominant Brachydactyly type B (BDB), further emphasizing critical functions of Ror2 during developmental morphogenesis. In this article, we also discuss the signaling machinery mediated by Ror-family RTKs with a particular emphasis on our recent structure-function analyses of Ror-family RTKs.     

An expression pattern screen for genes involved in the induction of the posterior nervous system of zebrafish

The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.     

Defect in cell wall integrity of the yeast saccharomyces cerevisiae caused by a mutation of the GDP-mannose pyrophosphorylase gene VIG9

The Saccharomyces cerevisiae VIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate. Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity. During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability. JS30 excreted cell surface proteins in the medium without any indication of cell lysis. Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene. These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity.     

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.