Over-expression of ζ glutathione S-transferase in transgenic rice enhances germination and growth at low temperature

To develop a rice cultivar that would be suitable for direct-seedingcultivation in cooler temperate regions, we generated transgenic rice plants inwhich a rice encoding a -class glutathioneS-transferase (GST) under the control of a maize ubiquitinpromoter. GSTs have been suggested to be responsible for tolerance to variousstresses such as cold, salt and drought by detoxification of xenobioticcompounds and reactive oxygen species. A total of 87 R₀ transgenicrice plants harboring a chimeric GST gene were generatedusing Agrobacterium mediated transformation. ThreeR₂ lines homozygous for the transgene were assayed for GST activityand had higher GST and glutathione peroxidase activities thannon-transformants.Seedlings of the transgenic lines demonstrated greatly enhanced germination andgrowth rates at low temperature grown under submergence. The GST transgeniclines should be useful for breeding rice cultivars suitable for direct-seedingcultivation in cooler temperate regions.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Azelastine and suplatast shorten the distribution half-life of IgE in rats

We aim to clarify whether suplatast and azelastine (anti-allergic drugs) can shorten the half-life of imnunoglobulin E (IgE) in the circulating blood. Thirty Wistar rats were divided into six groups. Distilled water or anti-allergic drugs were given orally for 6 days after the first sensitization. Two milligrams of monoclonal dinitrophenyl (DNP)-specific rat IgE was administered to the rats, which had been given suplatast or azelastine orally. The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. The elimination half-life of rat IgE was about 12 h irrespective of the sensitized state. The intercompartmental rate constants (Kct and Ktc) in the suplatast-administered or azelastine-administered group were larger than those of the distilled water-administered group under non-sensitized conditions. These findings suggested that the anti-allergic drugs used in the present study facilitated the excretion of IgE from the circulation in rats.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.     

Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.     

Production of hydroxyl radicals and alpha-dicarbonyl compounds associated with Amadori compound-Cu2+ complex degradation

The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1-n-butylamino-D-fructose (DBF) and N(alpha)-formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu2+ (but no other transition metal ions) to form a DBF-Cu2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu+ during incubation at 37 degrees C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and alpha-dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and a-dicarbonyl compounds from Amadori adducts in the presence of Cu2+.     

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.     

Microbial community changes during organic solid waste treatment analyzed by double gradient-denaturing gradient gel electrophoresis and fluorescence in situ hybridization

The bacterial community present during semicontinuous treatment of organic solid waste under alkaline and high-temperature conditions was studied. PCR-amplified 16S rDNA fragments were analyzed by double gradient-denaturing gradient gel electrophoresis (DGGE). The band pattern was stable during the steady state of the treatment phase, and the major bands resulting from individual treatments had the same DNA sequence with good reproducibility. No sequence in the DNA database of isolated bacteria showed close similarity to this sequence, the closest relative being Bacillus licheniformis with less than 97% similarity. The conditions for fluorescence in situ hybridization (FISH) were determined without the need to obtain extracts of the bacterial cells. An oligonucleotide probe was designed to detect the microorganisms found in the DGGE analysis. FISH analysis showed that the bacterium corresponding to the major bands accounted for 30% of the total eubacterial cell count at the steady state. These results indicate that this bacterium is a key microorganism in the biodegradation process.     

Role of glutamine and arginase in protection against ammonia-induced cell death in gastric epithelial cells

Ammonia is a cytotoxic factor produced during Helicobacter pylori infection that may reduce the survival of surface epithelial cells. Here we examine whether ammonia kills cells and whether L-glutamine (L-Gln) protects against cell death by stimulating ammonia detoxification pathways. Cell viability and vacuolation were quantified in rat gastric epithelial (RGM1) cells incubated with ammonium chloride at pH 7.4 in the presence or absence of L-Gln. Incubation of RGM1 cells with ammonium chloride caused a dose-dependent increase in cell death and vacuolation, which were both inhibited by L-Gln. We show that RGM1 cells metabolize ammonia to urea via arginase, a process that is stimulated by L-Gln and results in reduced ammonia cytotoxicity. L-Gln also inhibits the uptake and facilitates the extrusion of ammonia from cells. Blockade of glutamine synthetase did not reduce the survival of RGM1 cells, demonstrating that the conversion of L-glutamate and ammonia to L-Gln is not involved in ammonia detoxification. Thus our data support a role for L-Gln and arginase in protection against ammonia-induced cell death in gastric epithelial cells.