A novel methodology for characterizing strand-break termini and damaged bases in plasmid DNA exposed to ionizing radiation

We have developed a de novo methodology to characterize radiation damage in DNA. An enzyme system consisting of the 3'-->5' exonuclease snake venom phosphodiesterase (SVPD) and calf intestine alkaline phosphatase (CIAP) was used to examine the 3' termini of strand-break sites. In this study, we hypothesized that the strand-break termini can be divided into two categories: CIAP-independent SVPD sites and CIAP-dependent SVPD sites. The former consists of strand-break termini that can be recognized directly and digested by SVPD without CIAP pretreatment, whereas the latter includes the termini that cannot be digested by SVPD without CIAP pretreatment. In addition, the apparent radiation-chemical yield (G value) can be estimated using the level of intact 2'-deoxynucleotides produced during a 15-min incubation with SVPD. The G value for total strand breaks in fully dried DNA irradiated with (60)Co gamma-rays was estimated to be 0.1 micromol/J. Moreover, the G values of CIAP-dependent and CIAP-independent SVPD sites were estimated to be 0.078 and 0.024 micromol/J, respectively. These values suggest that 3'-phosphate termini are more likely to be produced than 3' termini without phosphate. Furthermore, piperidine-treated irradiated plasmid DNA was also treated with the same enzyme system to examine the piperidine-labile sites. As a result of the treatment, the G value of the CIAP-dependent SVPD sites increased to 0.16 micromol/J, whereas no significant increase was seen in the G value of the CIAP-independent SVPD sites. This observation implies that most piperidine-labile damaged bases can be eliminated to form apurinic/apyrimidinic sites, which are completely removed by piperidine treatment to form 3' phosphate termini, and that prompt CIAP-independent SVPD sites are piperidine resistant.     

Yangtze finless porpoises along the main channel of Poyang Lake,China: Implications for conservation

Poyang Lake is the largest freshwater lake in China and flows into the Yangtze River. It is a traditional habitat for the endangered Yangtze finless porpoise, which has not been well investigated. To reveal the distribution of the porpoise in Poyang Lake, 12 passive acoustic surveys were conducted along 123 km of the main channel of the lake during different seasons (spring transition season, wet season, autumn transition season, and dry season) from 2008 to 2012. We counted the number of phonating porpoises encountered and calculated the detection rate (encountered individuals detected per kilometer). The median porpoise detection rates ranged from 0 to 0.65 individuals per kilometer during the different surveys. The highest median detection rate of 0.50 was detected in the autumn transition season. The seasonal shrinking of the lake during the dry season may cause a concentration of porpoises in the narrow channels and a high incidence of collisions with cargo ships and fishing boats. Conservation actions should be focused on the main channel of the lake during the dry and transition seasons. In addition, the expansion of the existing reserve to include areas with high porpoise detection rates is necessary.     

Larvicidal activity of (-)-dihydroguaiaretic acid derivatives against Culex pipiens

The larvicidal activity against Culex pipiens of all stereoisomers of dihydroguaiaretic acid (DGA) and secoisolariciresinol was measured, and these DGAs were found to be potent. Sixteen (-)-DGA derivatives were then newly synthesized to analyze their structure-activity relationship. Two derivatives monohydroxylated at the 3- or 4-position of the 7-phenyl group of DGA induced acute paralytic activity in the mosquitoes. Derivatives with several hydroxyl groups had lower activity than the natural compound, suggesting that hydrophobicity was probably an important factor for their insecticidal activity.     

αvβ3-Integrin-targeting lanthanide complex: synthesis and evaluation as a tumor-homing luminescent probe

The application of lanthanide complexes in the time-resolved fluorescence imaging of living cells has emerged in the last few decades, providing high-contrast images of cells through detection of the delayed emission. In the present study, we synthesized novel trivalent lanthanide complexes containing the cyclic peptide c(RGDfK) to visualize the α_vβ₃-integrin-expressing tumor cells. Conjugation of c(RGDfK) with the macrocyclic bipyridine ligand had little effect on the fluorescence properties of the complex, indicating that the coordinated lanthanide ion was well isolated from the peptide. Bright luminescence images of α_vβ₃-integrin-expressing U87-MG cells were successfully obtained by employing the probes.     

Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.     

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.     

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.     

Inhibitors of human 2,3-oxidosqualene cyclase (OSC) discovered by virtual screening

Five human 2,3-oxidosqualnene cyclase (OSC) inhibitors were discovered using the combination of a virtual screening based on a docking study and an isotope-free assay system. All of these inhibitors were revealed to have activities comparable or superior to that of LDAO, a known OSC inhibitor. The computational study of the newly identified inhibitors suggested that CH/π interactions with F444 and W581 contribute to the binding, and these interactions are candidates for additional structural filters in the next generation of virtual screening.     

Differences in amounts of pools and riffles between upper and lower reaches of a fully sedimented dam in a mountain gravel-bed river

To understand the extent of changes in the habitat structure of macroinvertebrates due to channel degradation, the amount of pools and riffles and bed materials were determined in upper and lower reaches of a fully sedimented dam in a mountain gravel-bed river. Pools occurred more frequently and individual pools were longer in the lower reach than in the upper reaches; the total length of pools in the former was four times than in the latter. In contrast, individual riffles in the upper reaches were longer than and the total length double that in the lower reach. The main bed materials in the upper reaches were moderate-sized cobbles and boulders (diameter 10–40 cm), which generated moderately steep and long riffles. In the lower reach, these materials were scarce and the main bed materials were large boulders (>50 cm) and bedrock, which generated steep and short riffles; finer materials (<10 cm) dominated in pools and runs. The reduced total length of riffles in the lower reach was partly associated with the steepening of individual riffles, which may be due to the shortage of moderate-sized cobbles and boulders. Macroinvertebrate biomass at reach scale, which was estimated based on the total lengths of pools and riffles, was more than two to four times in the upper reaches than in the lower reach.