Survival of genetically modified and self-cloned strains of commercial baker's yeast in simulated natural environments: environmental risk assessment

Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (ATH1) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study, ATH1 disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains ATH1 loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of ATH1 by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.     

Ethnic differences in CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) genotypes in Japanese and Israeli populations

CYP2C9 is a major P450 2C enzyme, which hydroxylates about 16% of drugs that are in current clinical use and contributes to the metabolism of a number of clinically important substrate drugs such as warfarin. Ethnic differences in the genetic variation of CYP2C9 have been reported, and might be related to the frequencies of adverse reactions to drugs metabolized by CYP2C9 in different ethnic groups. In the present study, ethnic differences in the CYP2C9*2 and CYP2C9*3 allele distribution in Japanese and Israeli populations were evaluated using a newly developed oligonucleotide based DNA array (OligoArray(R)). The population studied consisted of 147 Japanese and 388 Israeli donors (100 Ashkenazi Jews, 99 Yemenite Jews, 100 Moroccan Jews and 89 Libyan Jews). The CYP2C9*2 [Arg144Cys (416 C>T), exon 3] and CYP2C9*3 [Ile359Leu (1061 A>C), exon 7] genotypes were determined using an OligoArray(R). The accuracy of genotyping by the OligoArray(R) was verified by the fluorescent dye-terminator cycle sequencing method. A Hardy-Weinberg test indicated equilibrium (chi(2)<3.84 is Hardy-Weinberg) in all populations. The CYP2C9*2 genotype (CC/CT+TT) was absent in Japanese (1/0) (OR 0.02), and its frequency was significant in Libyan Jews (0.697/0.303) (OR 2.13; 95% CI 1.07-4.24) compared with Ashkenazi Jews (0.83/0.17), Yemenite Jews (0.899/0.101), and Moroccan Jews (0.81/0.19). The frequencies of CYP2C9*3 genotype (AA/AC+CC) was significantly lower in Japanese (0.986/0.014) (OR 0.08), and was higher in Libyan Jews (0.652/0.348) (OR 3.03; 95% CI 1.5-6.1) and Moroccan Jews (0.77/0.23) (OR 1.69; 95% CI 0.62-3.48) compared with those in Ashkenazi Jews (0.85/0.15) and Yemenite Jews (0.849/0.151). Thus, the CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) variants were rare in the Japanese population, and showed different frequencies in the four Jewish ethnic groups examined.     

Using process diagrams for the graphical representation of biological networks

With the increased interest in understanding biological networks, such as protein-protein interaction networks and gene regulatory networks, methods for representing and communicating such networks in both human- and machine-readable form have become increasingly important. Although there has been significant progress in machine-readable representation of networks, as exemplified by the Systems Biology Mark-up Language (SBML) (http://www.sbml.org) issues in human-readable representation have been largely ignored. This article discusses human-readable diagrammatic representations and proposes a set of notations that enhances the formality and richness of the information represented. The process diagram is a fully state transition-based diagram that can be translated into machine-readable forms such as SBML in a straightforward way. It is supported by CellDesigner, a diagrammatic network editing software (http://www.celldesigner.org/), and has been used to represent a variety of networks of various sizes (from only a few components to several hundred components).     

Discovery of diphenylcarbamate derivatives as highly potent and selective IP receptor agonists: orally active prostacyclin mimetics. Part 3

The new classes of diphenylcarbamate derivatives with a tetrahydronaphthalene skeleton as highly potent and selective IP agonists have been discovered. The optimized diphenylcarbamate type compound FK-788: (R)-4 exhibited potent antiaggregative potency with an IC50 of 18 nM and high binding affinity for the human recombinant IP receptor with K(i) values of 20 nM and selectivity for human IP over all other members of the human prostanoid receptor family. Compound (R)-4 was shown to exhibit good pharmacokinetic properties in rats and dogs, and also good bioavailability in healthy volunteers.     

Syntheses and properties of the major hydroxy metabolites in humans of blonanserin AD-5423, a novel antipsychotic agent

Two major metabolites in humans of blonanserin, 2-(4-ethyl-1-piperazinyl)-4-(4-fluorophenyl)-5,6,7,8,9,10-hexahydrocycloocta-[b]pyridine (code name AD-5423), were synthesized. The first, 7-hydroxylated AD-5423, was synthesized through a four-step process starting from 4-fluorobenzoylacetonitrile (1), and the second, 8-hydroxylated AD-5423, a nine-step process also from 1. The optical resolution, structures, and receptor binding properties of the metabolites were documented.     

Powerful skin cancer protection by a CPD-photolyase transgene

BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.     

Protein fishing with chiral molecular baits

Chirality plays a central role in various biological recognition processes. Here a methodology was developed to utilize chiral recognition processes for the selective biotinylation of proteins in crude cell lysates. Two pairs of diastereomeric probes containing benzophenone and biotin were prepared through solid-phase synthesis. Protein-binding selectivity of each probe was examined by photo-cross-linking of cell lysates, followed by SDS-PAGE and Western blot. The study revealed that our approach permits selective labeling of benzophenone-binding proteins in complex proteomes. In addition, it was found that the selectivity depends largely on a single chiral center and substitutions in the vicinity of benzophenone. Taken together, the current work demonstrates that chiral recognition process can be employed to selectively label proteins in complex proteomes. Thus the study opens up the possibility to expand the scope of chemical proteomics research for various applications, including biomarker discovery, drug screening and development.     

Synthesis and in vivo biodistribution of BPA-Gd-DTPA complex as a potential MRI contrast carrier for neutron capture therapy

p-boronophenylalanine (BPA) conjugated Gd-DTPA complex (3) was synthesized from the active methyne compound 6, the allylic carbonate 7, and BPA by the palladium-catalyzed allylation reaction followed by the DCC coupling reaction. The in vivo biodistribution of complex 3 was evaluated by prompt gamma-ray analysis and alpha-autoradiography using the tumor-bearing rats. High accumulation of gadolinium was observed in the kidney and the %ID values were 0.17 and 0.088 at 20 and 60 min after injection of 3, respectively. The accumulation was also observed in the tumor and the %ID values were 0.010 and 0.0025 at 20 and 60 min after injection, respectively. The visualization experiment of boron distribution in the tumor-bearing rat by alpha-autoradiography indicates that boron was accumulated in the tumor and the intestines at 20 min after injection.