Biotransformation of 1-Nitropyrene in Intestinal Anaerobic Bacteria

Mutagenic nitroaromatic compounds have recently been found in photocopies, urban atmosphere, automobile exhaust and wastewater. 1-Nitropyrene (1-NP) is readily formed when pyrene, ubiquitous in the environment, is exposed to nitrogen dioxide in the urban atmosphere or in automobile exhaust, and is highly mutagenic, inducing 449 his⁺ revertants/plate/nmol from Salmonella typhimurium strain TA98 in the absence of S9 fraction in the Salmonella-microsome test. It is possible to swallow sputum or some food containing 1-NP and it would come into contact with the normal bacterial flora. We determined the 1-NP nitroreductase activity in environmental and laboratory bacterial strains. We found that the mutagenicity of 1-NP mixed with the feces of a healthy man or a culture of anaerobic bacteria was decreased. The product proved to be 1-aminopyrene (1-AP), based on its fluorescence spectrum, its mass spectrum, and its characteristic thin layer chromatographic and high performance liquid chromatographic patterns. The 1-NP nitroreductase activity of aerobic bacteria was low, but crude extracts from the anaerobic bacteria, i.e., Bacteroides fragilis, B. thetaiotaomicron, B. vulgatus, Fusobacterium mortiferum, F. nucleatum, Clostridium perfringens, C. sporogenes, Bifidobacterium adolescentis, B. bifidum, Eubacterium lentum, E. limosum, and Peptostreptococcus anaerobius, all easily converted 1-NP to 1-AP and proportionally decreased the mutagenic activity of 1-NP.     

A reliable measure of similarity based on dependency for short time series: an application to gene expression networks

Background  

Microarray techniques have become an important tool to the investigation of genetic relationships and the assignment of different phenotypes. Since microarrays are still very expensive, most of the experiments are performed with small samples. This paper introduces a method to quantify dependency between data series composed of few sample points. The method is used to construct gene co-expression subnetworks of highly significant edges.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Generation and characterization of monoclonal antibodies that specifically recognize p65/L-plastin isoform but not T-plastin isoform

The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca(2+)-, calmodulin-, and beta-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.     

Effects of dietary oyster extract on lipid metabolism, blood pressure, and blood glucose in SD rats, hypertensive rats, and diabetic rats

Oyster extract was prepared by hydrolysis of oyster protein with proteases, Aloase (a protease from Bacillus subtilis), and Pancitase (a protease from Aspergillus oryzae). Rats were fed a diet containing 20% casein (the control diet) or 15% casein and 5% oyster extract (the oyster extract diet) as the protein source. The oyster extract diet exerted a significant reduction in serum cholesterol and liver triglyceride concentrations as compared with the control diet in Sprague-Dawley (SD) rats fed cholesterol-supplemented diets for 4 weeks. The activities of cytosolic fatty acid synthase and glucose-6-phosphate dehydrogenase were significantly lower in the oyster extract group than in the control group in the liver of SD rats. Hepatic cholesterol and triglyceride concentrations were significantly lower in spontaneously hypertensive (SH) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, type 2 diabetic rats, fed the oyster extract diet, for 4 weeks and 4 months respectively, than in those fed the control diet in the cholesterol-free diet. Blood pressure was significantly lower in the oyster extract group than in the control group at the 2nd and 4th weeks after the beginning of feeding experimental diets in SH rats. These results suggest that oyster extract prepared by hydrolysis of oyster induces triglyceride-lowering activity in the liver through a decrease in hepatic lipogenesis in SD rats, and that it exerts the antihypertensive effect in SH rats.     

A Blue-shifted Light-driven Proton Pump for Neural Silencing

Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (∼570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (λ_max = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the β-ionone ring of the chromophore in the mutant is rotated ∼130° because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the π conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.     

Additional effects of silver nanoparticles on bactericidal efficiency depend on calcination temperature and dip-coating speed

There is an increasing interest in the application of photocatalytic properties for disinfection of surfaces, air, and water. Titanium dioxide is widely used as a photocatalyst, and the addition of silver reportedly enhances its bactericidal action. However, the synergy of silver nanoparticles and TiO(2) is not well understood. The photocatalytic elimination of Bacillus atrophaeus was examined under different calcination temperatures, dip-coating speeds, and ratios of TiO(2), SiO(2), and Ag to identify optimal production conditions for the production of TiO(2)- and/or TiO(2)/Ag-coated glass for surface disinfection. Photocatalytic disinfection of pure TiO(2) or TiO(2) plus Ag nanoparticles was dependent primarily on the calcination temperature. The antibacterial activity of TiO(2) films was optimal with a high dip-coating speed and high calcination temperature (600°C). Maximal bacterial inactivation using TiO(2)/Ag-coated glass was also observed following high-speed dip coating but with a low calcination temperature (250°C). Scanning electron microscopy (SEM) showed that the Ag nanoparticles combined together at a high calcination temperature, leading to decreased antibacterial activity of TiO(2)/Ag films due to a smaller surface area of Ag nanoparticles. The presence of Ag enhanced the photocatalytic inactivation rate of TiO(2), producing a more pronounced effect with increasing levels of catalyst loading.     

Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.