Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3.

The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D-peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.     

The role of cAMP in flagellation of Salmonella typhimurium.

A mutational alteration either in adenylate cyclase (cya-) or in cyclic-3'5'-AMP (cAMP) receptor protein (crp-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation cfs, partially suppressing the cya- mutation, was identified among the revertants of cya-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli.

Of the more than 30 genes required for flagellar function, 6 are located between pyrC and ptsG on the Escherichia coli genetic man. This cluster of genes is called flagellar region I. Four-point transductional crosses were used to establish the position and order of the region I flagellar genes with respect to the outside markers ptsG and pyrC. Bacteriophage lambda-E. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. The properties of specific hybrids that carried the region I fla genes were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the lambda hybrids demonstrated the existence of at least six flagellar-specific cistrons. These directed the synthesis of polypeptides with the following apparent molecular weights: flaV, 11,000; flaK, 42,000; flaL, 30,000 and 27,000; flaM, 38,000; flS, 60,000; and flaT, 35,000. Plasmid ColE1-E. coli hybrids with region I flagellar genes were also used to program the synthesis of polypeptides in minicell-producing strains. The polypeptides synthesized in these experiments were identical to polypeptides of the hook-basal body structure and helped to confirm the assignment of genes to specific polypeptides. The synthesis of all of these polypeptides was regulated by the same mechanism that regulates the synthesis of other flagellar-related structural components.     

Three new asymmetric trans-amine(azole)dichloridoplatinum complexes that overcome cisplatin resistance and their reactions with 5'-GMP

Three new asymmetric platinum(II) complexes comprising an isopropylamine ligand trans to an azole ligand were synthesized and fully characterized by ¹H NMR, ¹⁹⁵Pt NMR, IR and elemental analysis. In addition the X-ray crystal structure of all three complexes was determined. The reaction kinetics of the complexes with DNA model base guanosine-5′-monophosphate (GMP) was studied, revealing reaction kinetics comparable to cisplatin. To gain insight in the complexes as potential antitumor agents, cytotoxicity assays were performed on a variety of human tumor cell lines. These assays showed the complexes all to possess cytotoxicity profiles comparable to cisplatin. Furthermore, the complexes largely retain their activity in a human ovarian carcinoma cell line resistant to cisplatin, A2780R, compared to the cisplatin sensitive parent cell line A2780. These results are of fundamental importance, illustrating how platinum complexes of trans geometry can show improved activity compared to cisplatin in both cisplatin sensitive and cisplatin resistant cell lines.     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion     

A novel mutation in KNOPF uncovers the role of alpha-glucosidase I during post-embryonic development in Arabidopsis thaliana

N-glycosylation is a common protein modification. Joining of polypeptide and carbohydrate elements into hybrid molecules provides an opportunity to fine-tune protein properties. However, the role of N-glycosylation on the development of multicellular organisms remains elusive. Here we report a hypomorphic allele of KNOPF/GLUCOSIDASE 1, which allows us to describe the effects of impaired alpha-glucosidase I on post-embryonic development of plants for the first time. This knf-101 mutation alters cell shape but does not affect cell arrangements, except for the patterning of specialized epidermal cells, delineating the significance of N-glycan processing during epidermal development in Arabidopsis.     

Cyclosporin-A potently induces highly cardiogenic progenitors from embryonic stem cells

Though cardiac progenitor cells should be a suitable material for cardiac regeneration, efficient ways to induce cardiac progenitors from embryonic stem (ES) cells have not been established. Extending our systematic cardiovascular differentiation method of ES cells, here we show efficient and specific expansion of cardiomyocytes and highly cardiogenic progenitors from ES cells. An immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes by 10-20 times. Approximately 200 cardiomyocytes could be induced from one mouse ES cell using this method. Expanded progenitors successfully integrated into scar tissue of infracted heart as cardiomyocytes after cell transplantation to rat myocardial infarction model. CSA elicited specific induction of cardiac lineage from mesoderm in a novel mesoderm-specific, NFAT independent fashion. This simple but efficient differentiation technology would be extended to induce pluripotent stem (iPS) cells and broadly contribute to cardiac regeneration.