南沙链霉菌来源细胞色素P450酶CYP154C34的表征及生物转化

【目的】P450酶作为一种多功能生物催化剂,可在温和条件下高区域和立体选择性地催化复杂化合物中未活化的C-H键,因此P450酶在化工原料合成、环境污染物降解及药物合成等领域都具有重要作用。本文对南沙链霉菌基因组中的一个新颖的P450酶CYP154C34进行研究,通过构建异源表达和全细胞生物转化重组菌探究其功能。【方法】构建2种全细胞生物转化BL21(DE3)重组菌(含p ET28a-CYP154C34-RhFRED和pET28a-CYP154C34+pACYCDuet-Pdx/PdR)和1种异源表达BL21(DE3)重组菌(含pET28a-CYP154C34)。通过全细胞生物转化的方式筛选底物,分析催化功能及产物结构。比较2种全细胞生物转化重组菌和体外酶反应对底物的转化率。分析CYP154C34和不同底物及底物类似物的亲和力。【结果】通过底物筛选和产物鉴定发现CYP154C34可催化包括孕酮、睾酮、雄烯二酮在内的9种甾体化合物16α位羟基化。通过2种不同还原伴侣的全细胞体系及体外酶反应对底物转化率的比较,发现含有pET28a-CYP154C34-RhFRED的BL21(DE3)重组菌的...     

Continuous treatment with recombinant Mycobacterium tuberculosis CFP-10-ESAT-6 protein activated human monocyte while deactivated LPS-stimulated macrophage

Influence of the recombinant culture filtered protein 10 (CFP-10) and early-secreted antigenic target 6kDa protein (ESAT-6) (r-CFP-10-ESAT-6, rCE) of Mycobacterium tuberculosis (Mtb) on human monocyte and macrophage activation was investigated using human monocyte, monocyte like THP-1 cell line and monocyte derived macrophage (MDM). rCE solely enhanced TNF-alpha release from human monocytes and THP-1 cells in a dose- and time-dependent manner. rCE enhanced expression of CD80 and CD40, it also synergized with IFN-gamma in induction of TNF-alpha production and HLA-DR expression. Pharmacological agents that selectively inhibit mitogen activated protein kinase activation markedly suppressed rCE-induced TNF- alpha release. However, continuous presence of rCE (>72h) during monocyte to macrophage differentiation inhibited macrophage response to LPS stimulation. Collectively, these data suggest that rCE might have differential influence on monocyte and macrophage activation, which might be correlated with Mtb immune evasion.     

褐煤腐植酸对土壤氨氧化古菌群落结构的影响

【目的】研究腐植酸(HA)对土壤氨氧化古菌(AOA)的影响,进而探讨HA对土壤氮循环的作用。【方法】采用末端标记限制性多态性分析(T-RFLP)和实时定量PCR技术,研究了两种腐植酸(原生腐植酸-cHA和降解后的腐植酸-bHA)与尿素一同施加于土壤中的氨氧化古菌(AOA)和古菌的群落结构及数量的变化。【结果】只加尿素的处理AOA数量明显增加,其群落结构也发生明显变化,而加入尿素和两种腐植酸(HA)的处理土壤中,AOA数量增加得到明显的抑制,且典范对应分析(canonical correspondence analysis,CCA)表明尿素是影响AOA群落结构的最大因素,而HA可以缓冲尿素对AOA群落结构的影响,从而可以稳定AOA的群落结构。只加入尿素的处理还导致了古菌数量降低,而两种HA均抑制古菌数量的降低,表明HA可以缓冲尿素对古菌的影响。CCA分析表明时间是影响古菌群落结构的最重要因素,将时间作为共变量的部分典范对应分析(partial canonical correspondence analysis,pCCA)表明除时间外古菌的群落结构对cHA也比较敏感。【结论】这些结果表明HA通过抑制AOA数量而调控其与植物竞争氨来减少氨的损失,从而提高尿素利用率。     

Technology on sleep surgery

Sleep and Biological Rhythms - Sleep-disordered breating (SDB), including obstructive sleep apnea–hypopnea syndrome (OSAS) is a clinical syndrome mainly characterized by recurrent upper...     

A Time-Course Proteomic Analysis of Rice Triggered by Plant Activator BTH

Benzothiadiazole (BTH) is an artificial inducer of systemic acquired resistance. Due to rice being an important food crop and model plant, we investigated its response to BTH using label-free proteomics technology coupled with bioinformatics. Protein expression levels were verified using the multi-reaction monitoring mode and semi-quantitative RT-PCR. BTH treatment can up- or down-regulate many proteins produced by the rice host at all four periods, with the numbers of proteins being 6/24, 9/10, 14/10, and 8/20, respectively. Compared with mock treatments (phosphate buffered saline with 0.1 % dimethylsulfoxide and 0.5 % Tween-20), some proteins related to plant resistance were only detected after BTH treatments, such as ascorbate peroxidase (POD) 3, chitinase A, thioredoxin-dependent POD 2, beta-1,3-glucanase 2, POD superfamily protein, major facilitator superfamily (MFS) protein, copper/zinc-superoxide dismutase (SOD) 1, pathogenesis-related protein (PR) 1. Other proteins showing up-regulation after BHT treatment included PR-5, glyceraldehyde-3-phosphate dehydrogenase C, plasma-membrane associated cation-binding protein 1, and oxidoreductase family proteins. These results indicated that BTH was involved with inducing rice resistance. Some up-regulated proteins were also involved in other metabolic processes. The activity and expression level of POD, phenylalanine ammonia-lyase (PAL), and SOD, lipoxygenase (LOX), beta-1,3-glucanases, and chitinases were determined using the enzyme activity assay and semi-quantitative RT-PCR. These results indicated that BTH can enhance the activity of beta-1,3-glucanases, LOX, PAL, and POD. BTH can also induce up-regulation of the copper/zinc-SOD, ascorbate POD, glutathione POD 1, Chitinase, and LOX1 genes.     

XRCC1 Arg399Gln genetic polymorphism and the risk of hepatocellular carcinoma: a meta-analysis

The X-ray repair cross-complementing group 1 (XRCC1) gene, one of over 20 genes that participate in the base excision repair pathway, is thought to account for differences in susceptibility to hepatocellular carcinoma. To assess the relationship between the XRCC1 Arg399Gln polymorphism and the risk of hepatocellular carcinoma (HCC), we performed a meta-analysis. All the relevant studies were extracted from PubMed, Embase, the Chinese biomedicine databases, the Chinese national knowledge infrastructure, and the Wanfang databases (prior to August 2012). The meta-analysis was performed using all eligible studies, which covered a total of 2,554 cases and 3,320 controls, to examine the association between XRCC1 Arg399Gln polymorphism and the risk of HCC. Our analysis suggested that the variant genotypes of the XRCC1 Arg399Gln gene were associated with a significantly increased risk of HCC in a co-dominant model (Arg/Gln vs. Arg/Arg, odd ratios [OR] 1.39, 95 % confidence interval [CI] 1.08–1.79; Gln/Gln vs. Arg/Arg, OR 1.26, 95 % CI 1.04–1.52) and a dominant model (Arg/Gln + Gln/Gln vs. Arg/Arg OR 1.36, 95 % CI 1.07–1.72), whereas no association was observed in the recessive model (Gln/Gln vs. Arg/Gln + Arg/Arg, OR 1.05, 95 % CI 0.91–1.21). The results of the subgroup analysis by ethnicity indicated that the XRCC1 Arg399Gln polymorphism was associated with increased risk of HCC in Asian populations using the co-dominant model (Arg/Gln vs. Arg/Arg, OR 1.41, 95 % CI 1.06–1.87) and the dominant model (Gln/Gln vs. Arg/Gln + Arg/Arg, OR 1.35, 95 % CI 1.03–1.76). Our analysis provides evidence that the XRCC1 Arg399Gln polymorphism may be associated with a higher risk of HCC, especially among Asian populations.     

Systematic identification and characterization of miRNAs and piRNAs from porcine testes

microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) execute important regulatory roles in testis development and spermatogenesis, while previous studies mainly focus on the expression profiles in immature and mature porcine testes, which may cause a bottleneck for further understanding their complex physiological processes in porcine testes development and spermatogenesis. Thus, we presented the expression and characterization of miRNAs and piRNAs in DS (60-day-old), DN (90-day-old), DT (120-day-old) and DF (150-day-old) pig testes. In total, 12,834,628, 13,359,726, 12,851,249 and 12,938,601 clean reads were generated from these libraries, respectively. 293 mature and 36 novel miRNAs as well as 4923 piRNA clusters were identified from pig testes, and they showed an age-dependent manner. GO enrichment analysis of miRNA target genes and piRNA generated genes showed that they participated widely in regulating the pig spermatogenesis process. In addition, 12 differentially expressed miRNAs were randomly selected to validate using qRT-PCR. Our results provided novel comprehensive expression profiles of miRNAs and piRNAs in pig testes at different stages of sexual maturity, which will promote our knowledge of them in regulating the pig testes development and spermatogenesis process.     

Dose-Effects of Aorta-Infused Clenbuterol on Spinal Cord Ischemia-Reperfusion Injury in Rabbits

Background

The β₂ adrenergic receptor (β₂AR) plays an important role in ischemia-reperfusion (I/R) injury in various organs. Recently, a selective β₂AR agonist clenbuterol was suggested to protect against cerebral I/R injury. This study was designed to investigate changes of β₂ARs after spinal cord I/R injury and dose-effects of aorta-infused clenbuterol on spinal cord I/R injury in rabbits.

Methods

Spinal cord ischemia was induced in New Zealand white rabbits by infrarenal abdominal aortic occlusion with a balloon catheter for 30 minutes except the sham group. During occlusion, nothing (I/R group), normal saline (NS group) or clenbuterol at different doses of 0.005, 0.01, 0.05, 0.1, 0.5, or 1 mg/kg (C_0.005, C_0.01, C_0.05, C_0.1, C_0.5, and C₁ groups) was infused into the occluded aortic segments. The hemodynamic data, blood glucose and serum electrolytes were measured during experimental period. Neurological function was assessed according to the modified Tarlov scales until 48 hours after reperfusion. After that, the lumbar spinal cord was harvested for β₂AR immunohistochemistry and histopathologic evaluation in the anterior horns.

Results

The β₂AR expression in the anterior horns of the spinal cord was significantly higher in the I/R group than in the sham group. Tarlov scores and the number of viable α-motor neurons were higher in C_0.01-C_0.5 groups than in the NS group, C_0.005 and C₁ groups and were highest in the C_0.1 group. Hypotension and hyperglycemia were found in the C₁ group.

Conclusion

β₂ARs in the anterior horn were upregulated after spinal cord I/R injury. Aortic-infused clenbuterol (0.01–0.5 mg/kg) can attenuate spinal cord I/R injury dose-dependently during the ischemic period. The Optimal dosage was 0.1 mg/kg. Activation of β₂AR could be a new therapeutic strategy for the treatment of spinal cord I/R injury.     

A Novel B-Cell Epitope Identified within Mycobacterium tuberculosis CFP10/ESAT-6 Protein

Background

The 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1∶1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown.

Methods

In the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera.

Results

One linear B-cell epitope (KWDAT) consistent with the 162^nd–166^th sequence of CE and the 57^th–61^st sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals.

Conclusion

There was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.