Light-induced DNA cleavage by esperamicin and neocarzinostatin

Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min.     

A novel system combining biocatalytic dephosphorylation of L-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.     

Learning genetic regulatory network connectivity from time series data

Recent experimental advances facilitate the collection of time series data that indicate which genes in a cell are expressed. This information can be used to understand the genetic regulatory network that generates the data. Typically, Bayesian analysis approaches are applied which neglect the time series nature of the experimental data, have difficulty in determining the direction of causality, and do not perform well on networks with tight feedback. To address these problems, this paper presents a method to learn genetic network connectivity which exploits the time series nature of experimental data to achieve better causal predictions. This method first breaks up the data into bins. Next, it determines an initial set of potential influence vectors for each gene based upon the probability of the gene's expression increasing in the next time step. These vectors are then combined to form new vectors with better scores. Finally, these influence vectors are competed against each other to determine the final influence vector for each gene. The result is a directed graph representation of the genetic network's repression and activation connections. Results are reported for several synthetic networks with tight feedback showing significant improvements in recall and runtime over Yu's dynamic Bayesian approach. Promising preliminary results are also reported for an analysis of experimental data for genes involved in the yeast cell cycle.     

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b⁺/⁻ mice

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.     

Complete genome sequence of highly multidrug-resistant Pseudomonas aeruginosa NCGM2.S1, a representative strain of a cluster endemic to Japan

We report the completely annotated genome sequence of Pseudomonas aeruginosa NCGM2.S1, a representative strain of a cluster endemic to Japan with a high level of resistance to carbapenem (MIC ≥ 128 μg/ml), amikacin (MIC ≥ 128 μg/ml), and fluoroquinolone (MIC ≥ 128 μg/ml).     

Development and characterization of a xylose-inducible gene expression system for Clostridium perfringens

A xylose-inducible gene expression vector for Clostridium perfringens was developed. Plasmid pXCH contains a chromosomal region from Clostridium difficile (xylR-P(xy)(lB)): xylR, encoding the xylose repressor, xylO, the xyl operator sequence, and P(xylB), the divergent promoter upstream of xylBA encoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study of C. perfringens.