Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

Changes in PAL,CHS, DAHP synthase (DS-Co and Ds-Mn) activity during anthocyanin synthesis in suspension culture of Fragaria ananassa

The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL), and chalcone synthase (CHS) was monitored at various light intensities (dark, 8.88 μmol m⁻² s⁻¹, 88.8 μmol m⁻² s⁻¹) using a strawberry cell suspension culture. DS-Mn, PAL, and CHS were found to increase significantly (p>0.05) under light intensitie of 88.8 μmol m⁻² s⁻¹ compared to those of 8.88 μmol m⁻² s⁻¹ and dark. The activity of DS-Mn, PAL, and CHS were maximum at 88.8 μmol m⁻² s⁻¹. Anthocyanin content reached a maximum after 48–60 h of culturing at 88.8 μmol m⁻² s⁻¹. DS-Co showed greater activity than DS-Mn during cell culturing, but showed no correlation with anthocyanin production and light intensity. The CHS gene expression was continuous at a light intensity of 88.8 μmol m⁻² s⁻¹. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis and antinephritic activities of quinoline-3-carboxamides and related compounds.

A series of linomide-related quinoline-3-carboxamides and their analogues was prepared and evaluated for antinephritic activities. The 6-MeS derivative 7a was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.     

Albutensin A and complement C3a decrease food intake in mice

Albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg) derived from serum albumin dose-dependently decreased food intake after intracerebroventricular (10-50 nmol/mouse) or peripheral (0.3-1.0 micromol/mouse) administration in fasted conscious ddY mice. Albutensin A delayed gastric emptying and elevated blood glucose levels. Although albutensin A showed low affinity for bombesin receptor, it decreased food intake in bombesin receptor knockout mice, indicating that its inhibitory effect on feeding was not mediated through bombesin receptor. Then, we investigated whether the albutensin A-induced decrease in food intake was mediated by complement C3a and C5a receptors, because albutensin A had affinities for these receptors. Des-Arg-albutensin A, lacking affinity for C3a and C5a receptors, did not inhibit food intake. We found for the first time that centrally administered C3a (10-100 pmol/mouse) by itself decreased food intake in fasted mice. In contrast, C5a increased food intake after central injection. Based on these results, we conclude that the inhibitory effect of albutensin A on food intake is mediated through the C3a receptor.     

Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.     

Crystal structure of Ufc1, the Ufm1-conjugating enzyme

Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.     

Production of different types of mannosylerythritol lipids as biosurfactants by the newly isolated yeast strains belonging to the genus Pseudozyma

Mannosylerythritol lipids (MEL), which are abundantly secreted by yeasts, are one of the most promising biosurfactants known. To obtain various types of MEL and to attain a broad range of applications for them, screening of novel producers was undertaken. Thirteen strains of yeasts were successfully isolated as potential MEL producers; they showed high production yields of MEL of around 20 g l(-1) from 40 g l(-1) of soybean oil. Based on the taxonomical study, all the strains were classified to be the genus Pseudozyma. It is interesting to note that they were categorized into three groups according to their production patterns of MEL. The first group, which included 11 strains taxonomically closely related to high-level MEL producers such as Pseudozyma antarctica and Pseudozyma aphidis, mainly produced 4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-A) together with 4-O-[(6'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-B) and 4-O-[(4'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl]-meso-erythritol (MEL-C) as the minor components. The second group of one strain, which was related to Pseudozyma tsukubaensis, predominantly produced MEL-B. The third group of one strain, which was closely related to Pseudozyma hubeiensis, mainly produced MEL-C; this is the first observation of the efficient production of MEL-C from soybean oil. Moreover, the major fatty acids of the obtained MEL-C were C(6), C(12), and C(16) acids, and were considerably different from those of the other MEL hitherto reported. The biosynthetic manner for MEL is thus likely to significantly vary among the Pseudozyma strains; the newly isolated strains would enable us to attain a large-scale production of MEL and to obtain various types of MEL with different hydrophobic structures.     

Activation of caspase 3 in HepG2 cells by elaidic acid (t18:1)

In order to examine the effects of trans-unsaturated fatty acids (TFAs) on HepG2 cells, cells were grown in serum-free media supplemented with elaidic acid (t18:1); t18:1 is the trans-isomer of oleic acid and is the major component of TFAs in foods. Both t18:1 and palmitic acids (16:0) at concentrations higher than 100 microM inhibited growth and decreased the rate of protein synthesis. The presence of phosphatidylserine in the outer leaflet of the lipid bilayer, indicative of apoptosis, occurred 1 h after the addition of both t18:1 and 16:0 to the media. Caspase 3 was found to be activated by these fatty acids: caspase 8 was activated by 16:0 and only moderately by t18:1. Activation of caspase 3 by these fatty acids was fully inhibited by a caspase 8 inhibitor. However, growth inhibition by t18:1 was partially prevented by the caspase 8 inhibitor. These results suggest that cell death caused by t18:1 may proceed by both caspase-dependent and -independent pathways.