Transcriptional Induction of Two Genes for CCaPs, Novel Cytosolic Proteins, in Arabidopsis thaliana in the Dark

Ca2+-signaling in downstream effectors is supported by many kinds of Ca2+-binding proteins, which function as a signal mediator and a Ca2+-buffering protein. We found in Arabidopsis thaliana a new type of Ca2+-binding protein, CCaP1, which consists of 152 amino acid residues, and binds (45)Ca2+ even in the presence of a high concentration of Mg2+. We found two other proteins with similar motifs, CCaP2 and CCaP3. These three proteins had no organelle localization signal and their green fluorescent protein (GFP) fusions were detected in the cytosol. Real-time PCR and histochemical analysis of promoter-beta-glucuronidase fusions revealed that CCaP1 was predominantly expressed in petioles while CCaP2 was expressed in roots. CCaP3 was hardly expressed. Expression of CCaP1 and CCaP2 was enhanced in darkness and became maximal after 24 h. Immunoblotting revealed petiole-specific accumulation of CCaP1. Expression of CCaP1 and CCaP2 was suppressed by a high concentration of Ca2+ and other metal ions. Deletion of sucrose from the medium markedly increased the mRNA levels of CCaP1 and CCaP2 within 2 h. Gibberellic acid enhanced the expression of CCaP1 and CCaP2 by 5- and 2.5-fold, respectively, after 6 h. CCaP1 and CCaP2 were suppressed in the petiole and the root, respectively, by light and the product of photosynthesis (sucrose) or both. These results suggest that CCaP1 functions as a mediator in response to continuous dark or gibberellic acid.     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.     

Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase

Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.     

Purification, cloning, and expression of an alpha/beta-galactoside alpha-2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

A novel sialyltransferase, alpha/beta-galactoside alpha-2,3-sialyltransferase, was purified from the cell lysate of a luminous marine bacterium, Photobacterium phosphoreum JT-ISH-467, isolated from the Japanese common squid (Todarodes pacificus). The gene encoding the enzyme was cloned from the genomic library of the bacterium using probes derived from the NH(2)-terminal and internal amino acid sequences. An open reading frame of 409 amino acids was identified, and the sequence had 32% identity with that of beta-galactoside alpha-2,6-sialyltrasferase in Photobacterium damselae JT0160. DNA fragments that encoded the full-length protein and a protein that lacked the sequence between the 2nd and 24th residues at the NH(2) terminus were amplified by polymerase chain reactions and cloned into an expression vector. The full-length and truncated proteins were expressed in Escherichia coli, producing active enzymes of 0.25 and 305 milliunits, respectively, per milliliter of the medium in the lysate of E. coli. The truncated enzyme was much more soluble without detergent than the full-length enzyme. The enzyme catalyzed the transfer of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid to disaccharides, such as lactose and N-acetyllactosamine, with low apparent K(m) and to monosaccharides, such as alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside, with much lower apparent K(m). Thus, this sialyltransferase is unique and should be very useful for achieving high productivity in E. coli with a wide substrate range.     

Molecular cloning, expression and properties of an alpha/beta-Galactoside alpha2,3-sialyltransferase from Vibrio sp. JT-FAJ-16

We cloned, expressed and characterized a novel alpha/beta-galactoside alpha2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a alpha2,3-sialyltransferase gene from a marine bacterium as a probe, a DNA sequence encoding a 402-amino-acid protein was identified from the JT-FAJ-16 genomic library. The protein showed 27.3-64.7% identity to the bacterial sialyltransferases classified into glycosyltransferase family 80. The protein showed sialyltransferase activity when expressed in Escherichia coli. The N-terminal truncated form of the enzyme was amplified in E. coli and its recovered activity was 215.7 unit/l culture medium. It was purified as a single band on SDS-PAGE through the three chromatographic steps. The specific activity of the purified recombinant enzyme reached 57.5 unit/mg protein. The alpha2,3sialylation was confirmed by (1)H- and (13)C-NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20 degrees C. Interestingly, the enzyme used both the alpha- and beta-anomers of galactosides as acceptors, suggesting that it can be described as an alpha/beta-galactoside alpha2,3-sialyltransferase. The enzyme had a wide range of acceptor substrate specificities. It transferred N-acetylneuraminic acid (NeuAc) to various monosaccharides and various oligosaccharides, and both N-linked and O-linked asialo-glycoprotein. These results suggest that the enzyme can be used as a powerful tool for the study for glycotechnology.     

Synthesis and antibacterial activities of new quinolone derivatives utilizing 1-azabicyclo[1.1.0]butane

The ring-opening reactions of 1-azabicyclo[1.1.0]butane 3 with thiols 6a-f gave 3-sulfenylazetidine derivatives 7a-f in 50-92% yields. Treatment of 3 with aromatic amines 11a-e and dibenzylamine 11f in the presence of Mg(ClO(4))(2) afforded the corresponding 3-aminoazetidine derivatives 12a-f in 24-53% yields. These azetidine derivatives were introduced into the C7 position of a quinolone nucleus 8 to afford the corresponding fluoroquinolones 9a-f and 13a-f in 21-83% yields. Some of them exhibited superior antibacterial activity against quinolone-susceptible MRSA in comparison with clinically used fluoroquinolones, such as levofloxacin, ciprofloxacin, and gatifloxacin.     

Ortho-azo substituted phenylboronic acids for colorimetric sugar sensors

The phenylboronic acids substituted with an azo group on the ortho-position show a significant change in UV-vis spectra upon sugar binding. A new mechanism for the spectral change of the dyes is proposed based on the formation and cleavage of B-N dative bond between boronic acid group and azo group.     

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.