Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea

Polysaccharides (5, 10, 25, 50 and 100 microg ml(-1)) from soybeans and soybeans fermented with Phellinus igniarius or Agrocybe cylindracea inhibited cytochrome P450 1A1, cytochrome P450 1A2 and cytochrome P450 2B1 activities in rat liver microsomes. The polysaccharides (5, 10 and 25 microg ml(-1)) also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity. The most potent inhibitors of cytochrome P450 isozymes and ornithine decarboxylase activities were the polysaccharides from soybeans fermented with Agrocybe cylindracea.     

Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniciproducens

Acetic acid is by-product from fermentation processes for producing succinic acid using Mannheimia succiniciproducens . To obtain pure succinic acid from the final fermentation broth, acetic acid was selectively removed based on the different extractability of succinic acid and acetic acid with pH using tri-n-octylamine (TOA) as extractant. When successive batch extractions were performed using 0.25 mol TOA kg(-1) dissolved in 1-octanol at pH 5, the mol ratio of succinic acid to acetic acid before extraction was 4.9 and the final ratio after the fourth batch was 9.4.     

Cloning, Expression, and Biological Function of a dTDP-Deoxyglucose Epimerase (gerF) Gene from Streptomyces sp. GERI-155

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules which has antimicrobial activities against gram-positive bacteria. The deoxyhexose biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-155 genome has now been isolated. Four orf were identified and a putative orf, supposed to code for the dTDP-deoxyglucose epimerase gene, was designated as gerF. gerF was expressed in E. coli using recombinant expression vector pHJ3. The recombinant protein expressed in a soluble form. The enzyme was purified by Ni-affinity column using imidazole buffer as eluents. The molecular mass of the expressed protein correlated with the predicted mass (36,000 Da) deduced from the cloned gene sequence data. The purified enzyme produced maltol from dTDP-4-keto-6-deoxyglucose and it was confirmed that the expressed protein was dTDP-deoxyglucose epimerase catalyzing epimerization of C-3 and C-5 or C-3 of dTDP-4-keto-6-deoxyglucose.     

The activities of antioxidant enzymes in response to oxidative stresses and hormones in paraquat-tolerant Rehmannia glutinosa plants

All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40 %. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93 % after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Nonconcerted evolution of histone 3 genes in a liverwort, Conocephalum conicum

To estimate the extent of genetic variation at the DNA level, the histone 3 (H3) genes were sequenced from single individual each from the three cryptic species recognized based on allozyme analyses, YFS, J and T types of Conocephalum conicum and two closely related species, C. japonicum and Marchantia polymorpha. Although the H3 genes are known to be highly conserved, the nucleotide diversities were 0.128, 0.109, 0.108, 0.049 and 0.034. These values are 30 to 100 times higher than that in Drosophila melanogaster (0.001). Besides, there were considerable differences in the position, length and number of introns among the loci of H3 genes. The observed high level of nucleotide diversities was explained by the fixation of many random mutations, and non-concerted evolution that resulted from low rates of unequal crossing-over and gene conversion probably due to the dispersed structure of H3 genes on genome in this species. The non-concerted evolutionary pattern was established by the analysis of phylogenetic tree and divergence rates. This study confirmed previous results suggesting that natural populations of liverwort maintains high extent of variation at DNA level.     

Recovery of ammonium lactate and removal of hardness from fermentation broth by nanofiltration

Nanofiltration (NF) was investigated as an alternative to desalting electrodialysis (ED) and ion exchange for the recovery of ammonium lactate from fermentation broth. Three commercial NF membranes, NF45, NF70, and NTR-729HF, were characterized with 50 mM NaCl, MgSO(4), and glucose solutions. NF45 membrane was selected because it showed the lowest rejection of monovalent ion, the highest rejection of divalent ion, and the highest rejection of nonpolar molecule. Effects of the operating pressure were investigated in a range of 100-400 psig, on the flux, lactate recovery, and glucose and magnesium removal from a real fermentation broth containing about 1.0 M of ammonium lactate. The flux and recovery rate increased linearly with the pressure. However, lactate rejection also increased with the pressure, lowering the recovery yield. More magnesium ions and glucose were rejected as the pressure was increased, and at 400 psig, for example, magnesium ion was almost completely rejected, highlighting the chance of obviating the necessity of ion exchange to remove hardness, by using NF instead of desalting ED. Membrane fouling was not so severe as expected, considering the complex nature and a rather high concentration of the fermentation broth treated.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.