Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.

Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 µM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2.

[Detection and analysis of bocavirus in hospitalized children with respiratory infection].

OBJECTIVE: To detect human bocavirus (HBoV) and investigate its genetic and evolutionary characteristics in children with acute respiratory infection in Tianjin, China. METHODS: A total of 1,259 samples of nasopharyngeal aspirates were collected from children with a confirmed diagnosis of acute respiratory infection between January and December, 2012. Viral nucleic acid was extracted, HBoV was detected by real-time quantitative PCR, and the gene segments of nucleocapsid protein of HBoV in positive samples were amplified by PCR. Several products were randomly selected and sequenced.The sequence obtained was compared with the known sequence of HBoV, and a phylogenetic analysis was performed. All the samples were examined to detect for other common respiratory tract viruses. RESULTS: Among the 1,259 samples, the positive rate of HBoV was 4.53% (57/1,259), and among the 57 samples with positive HBoV, 75% (43/57) were positive in children with an age of 6-36 months. The positive rate of HBoV in children peaked in summer (from June to August), and there was a mixed infection with other viruses. Sequence analysis was performed for the PCR products from 36 positive samples, and the presence of HBoV was confirmed, with a higher homology to the known sequence of HBoV. CONCLUSIONS: In Tianjin, acute respiratory infection in some children may be associated with HBoV infection, which is commonly seen in infants with an age of 6-36 months. The peak of HBoV infection occurs in summer. The phylogenetic analysis shows a high homology to the known sequence of HBoV, with few gene sequence variations.

[Effect of lactuside B on the expression of bcl-2 and bax mRNA and their protein in rats' cerebral cortex after cerebral ischemia-reperfusion injury].

This study is to investigate the effect of the major chemical composition in rhizome of Pterocypsela elata, lactuside B, on expression of bcl-2, bax mRNA and their protein in rats' cerebral cortex after cerebral ischemia-reperfusion injury. First, middle cerebral artery ischemia-reperfusion injury model was established, and each group was treated with the corresponding medicines. Animals were separately sacrificed at 24 h and 72 h. The brain infarct volumes were detected by TTC dye, bcl-2 and bax mRNA expression was checked by RT-PCR, and the proteins of bcl-2 and bax were explored by two-step immunohistochemistry in cerebral cortex of rats. Lactuside B can reduce brain infarct volume of cerebral cortex of rats, increase the expression of bcl-2 mRNA and decrease that of bax mRNA. Moreover, the ratio of bcl-2 to bax mRNA is higher in 12.5 and 25 mg kg(-1) dose group, respectively, which is significantly different from that of model group (P < 0.05 or P < 0.01). Generally, either 12.5 or 25 mg kg(-1) dose group is better than positive control medicine nimodipine (P < 0.05 or P < 0.01). In addition, the expression of bcl-2 and bax protein is consistent with their gene expression. Infarct volume and the ratio of bcl-2 to bax mRNA expression are significantly different (P < 0.05 or P < 0.01) between 72 h and 24 h group. The results demonstrated that lactuside B could play a good role in resisting cerebral ischemia by upregulating the expression of bcl-2 mRNA and protein and downregulating that of bax mRNA and protein.

Evaluation on pore structures of organosilicate thin films by grazing incidence small-angle X-ray scattering.

Nanoporous thin films fabricated by both a core-shell-shaped organic-inorganic hybrid sphere (octa(2,4-dinitrophenyl)silsesquioxane, ODNPSQ) and a four-leg-numbered surfactant (polyoxyethylene sorbitan monolaurate, Tween-20) for porogens in a higher molecular weight precursor (polyphenylsilsesquioxane, PPSQ) were characterized, respectively, by grazing incidence small-angle X-ray scattering (GISAXS), and the measured 2D GISAXS profiles were analyzed quantitatively by using a GISAXS formula based on the distorted wave Born approximation (DWBA). The fitted 2D GISAXS data show that the PPSQ porous thin films imprinted with ODNPSQ porogen exhibit sphere-shaped closed pores with the average pore size within a range of 1.18-3.12 nm and pore size distribution widths about 3.0 nm when the porogen loadings increase from 10 to 40 wt % and those imprinted with Tween-20 porogen give out an average pore size of 1.07-1.29 nm and pore size distribution widths about 2.0 nm with the porogen loading varying from 5 to 30 wt %. The nanoporous dielectric thin films imprinted with ODNPSQ porogen show a reducing to the molecular aggregation of porogens and significant antiphase separation behavior in the cross-linked matrix.

Cytoplasm estrogen receptor ß5 as an improved prognostic factor in thymoma and thymic carcinoma progression.

A number of previous studies have reported that sex steroid hormones, including estrogens, are involved in the regulation of the thymic function. The aim of the present study was to investigate the expression of estrogen receptor ß5 (ERß5) in thymic tumors and the correlation between ERß5 expression and thymoma biological characteristics. The expression levels of ERß5 in thymic epithelial tumors was evaluated in 103 patents using immunohistochemical staining and reverse transcription-quantitative polymerase chain reaction. In addition, an indirect immunofluorescence assay was performed to evaluate the ERß5 expression levels in the TC1889 and T1682 cell lines. The survival outcome was estimated using Kaplan-Meier plots. The results indicated that ERß5 expression was mainly located in the thymic tumor cell cytoplasm (87.37%; 90/103 cases) and overexpression was observed in thymic tumors compared with normal thymic tissues (P=0.001). Using the Kruskal-Wallis test, a statistically significant association was observed between cytoplasmic ERß5 (cERß5) expression and thymic tumor subtypes (P=0.024) and stages (P=0.003 and R=-0.376). The Kaplan-Meier plots revealed that cERß5 expression was significantly associated with improved overall and progression-free survival (P=0.008 and P=0.004, respectively). The present study suggested that overexpression of cERß5 may indicate an improved prognosis and may be involved in the underlying mechanism through which estrogen inhibits thymoma and thymic carcinoma development.

Lactuside B decreases aquaporin-4 and caspase-3 mRNA expression in the hippocampus and striatum following cerebral ischaemia-reperfusion injury in rats.

This study aimed to investigate the effects of lactuside B (LB) on aquaporin-4 (AQP4) and caspase-3 mRNA expression in the hippocampus and the striatum following cerebral ischaemia-reperfusion (I/R) injury in rats. Cerebral I/R injury was established in Sprague-Dawley rats by occluding the middle cerebral artery for 2 h and then inducing reperfusion. Rats in the I/R + LB groups were treated with various doses of LB following reperfusion. Neurological deficit scores and brain water content were obtained to determine the pharmacodynamics of LB. Reverse transcription polymerase chain reaction was performed to determine the expression levels of AQP4 and caspase-3 mRNA in the hippocampus and the striatum. The results of the present study indicate that LB decreased the neurological deficit scores and the brain water content. In the hippocampus, AQP4 and caspase-3 mRNA expression levels were significantly downregulated in the I/R + LB groups at 24 and 72 h following drug administration, compared with those in the I/R group (P<0.05). In the striatum, LB was also shown to significantly reduce AQP4 and caspase-3 mRNA expression levels at 24 and 72 h following drug administration, compared with those in the I/R group (P<0.05). The effects became stronger as the LB dose was increased. The most significant reductions in AQP4 and caspase-3 mRNA expression were noted in the I/R + LB 25 mg/kg and I/R + LB 50 mg/kg groups at 72 h following drug administration. The results of the present study show that LB is capable of significantly downregulating AQP4 and caspase-3 mRNA expression in the hippocampus and striatum following cerebral I/R injury in rats. The mechanism by which LB improved ischaemic brain injury may be associated with changes in AQP4 and caspase-3 mRNA expression in the hippocampus and the striatum.