Improved artemisinin accumulation in hairy root cultures of Artemisia annua by (22S, 23S)-homobrassinolide

When (22S, 23S)-homobrassinolide (SSHB) was added at 1 g l^–1 to hairy root cultures of Artemisia annua, the production of artemisinin reached to 14 mg l^–1, an increment of 57% over the control. SSHB treatment led concomitantly to an increased biomass production of 15 g l^–1. A stimulatory activity of SSHB on nucleic acids and soluble protein content in hairy roots was also observed at the growth stage.     

Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain

Down syndrome (DS) patients present with morphological abnormalities in brain development, leading to mental retardation. Given the importance of actin cytoskeleton to form the basis of various cell functions, the regulation of actin system is crucial during brain development. We therefore aimed to study the expression levels of actin binding proteins in fetal DS and control cortex. We evaluated the levels of eight actin binding proteins using the proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectroscopical identification of protein spots. In fetal DS brain we found a significant reduction of the actin-related protein complex 2/3 (Arp2/3) 20 kDa subunit and the coronin-like protein p57, which are involved in actin filament cross-linking and nucleation and capping of actin filaments. We conclude that deficient levels of these proteins may, at least partially, be involved in the dysgenesis of the brain in DS.     

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Nucleoside diphosphate kinases in mammalian signal transduction systems: recent development and perspective

The role of nucleoside diphosphate (NDP) kinase with special reference to mammalian signal transduction systems was described. The interaction between NDP kinases and G proteins was reevaluated in view of their protein structural information and its significance was extended further on the basis of recent findings obtained with small molecular weight G proteins such as Rad, menin, and Rac. Meanwhile, observations suggesting involvement of NDP kinases in the regulation of cell growth and differentiation led to the realization that NDP kinases may play a crucial role in receptor tyrosine kinase signal transduction systems. In fact, a number of experimental results, particularly obtained with PC12 cells, implicate that NDP kinases appear to regulate differentiation marker proteins and cell-cycle-associated proteins cooperatively. Consequently, we propose a hypothesis that NDP kinases might act like a molecular switch to determine the cell fate toward proliferation or differentiation in response to environmental signals.     

Physiological and pathological relevance of extracellular NM23/NDP kinases

The nm23 gene is overexpressed in many hematological malignancies and other neoplasms. Some tumor cell lines that overexpress NM23 secrete this protein into extracellular environment. In this study, we found that the serum concentration of NM23-H1 protein was significantly higher in patients with various hematological malignancies. The serum level of NM23-H1 protein was clinically useful as a prognostic factor in malignant lymphoma and acute myelogeneous leukemia (AML). The level of NM23-H1 protein in all of the normal serum samples examined was lower than 10 ng/mL, while those in the tumors varied from about 0 to 1000 ng/mL. Exogenously added NM23-H1 protein did not affect the growth or survival of various leukemia and lymphoma cell lines. However, NM23-H1 protein inhibited the survival of adherent normal peripheral blood mononuclear cells (PBMNC) at 100–1000 ng/mL, and slightly stimulated the survival of nonadherent PBMNC. These results suggest that the effect of NM23-H1 protein on normal PBMNC may be associated with a poor prognosis in hematological malignancies.     

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of -galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. -galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.