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.     

Single-molecule imaging of cooperative assembly of gamma-hemolysin on erythrocyte membranes

Single-molecule fluorescence imaging was used to investigate assembly of Staphylococcus aureus LukF and HS monomers into pore-forming oligomers (gamma-hemolysin) on erythrocyte membranes. We distinguished the hetero-oligomers from the monomers, as indicated by fluorescence resonance energy transfer between different dyes attached to monomeric subunits. The stoichiometry of LukF (donor) and HS (acceptor) subunits in oligomers was deduced from the acceptor emission intensities during energy transfer and by direct acceptor excitation, respectively. Based on populations of monomeric and oligomeric intermediates, we estimated 11 sequential equilibrium constants for the assembly pathway, beginning with membrane binding of monomers, proceeding through single pore oligomerization, and culminating in the formation of clusters of pores. Several stages are highly cooperative, critically enhancing the efficiency of assembly.     

Transaldolase/glucose-6-phosphate isomerase bifunctional enzyme and ribulokinase as factors to increase xylitol production from D-arabitol in Gluconobacter oxydans

Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH(2)-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors.     

Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterised by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. Recently, it has become evident that a noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway, which is related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. Here we isolate and functionally characterise a zebrafish homologue of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localisation of Dsh by downregulating levels of Dsh protein. Furthermore, we show that pk1 interacts genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway.     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Identification of a new gene responsible for the oxygen tolerance in aerobic life of Streptococcus mutans

Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.     

Covalent linkage of polyamines to peptidoglycan in Anaerovibrio lipolytica

Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of Anaerovibrio lipolytica, a strictly anaerobic bacterium. The peptidoglycan was degraded with the N-acetylmuramyl-L-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified as L-alanine-D-glutamic acid(alphacadaverine)gammameso-diaminopimelic acid (DAP)-D-alanine and L-alanine-D-glutamic acid(alphaspermidine)gammameso-DAP-D-alanine, respectively. The N(1)-amino group of spermidine was linked to the alpha-carboxyl group of the D-glutamic acid residue of peptide II.