非生物胁迫下耐辐射异常球菌drB0118基因功能分析

【目的】鉴定和确定被预测为编码干燥相关蛋白的耐辐射异常球菌(Deinococcus radiodurans) drB0118基因功能,探讨该基因对盐、渗透和氧化胁迫抗性的作用。【方法】构建drB0118基因缺失突变株(ΔB0118),通过氯化钠、D-山梨糖醇和过氧化氢等胁迫冲击实验及氧化胁迫条件下qRT-PCR分析,研究drB0118突变对非生物胁迫反应及氧化胁迫相关基因表达的影响。【结果】drB0118突变导致菌株对NaCl和D-sorbitol胁迫的抗性降低;对氧化胁迫(H2O2)敏感;qRT-PCR分析显示,drB0118突变引起氧化胁迫抗性基因pod和oxyR分别下调4倍和10倍。【结论】D. radiodurans中drB0118参与了盐、渗透和氧化等多种非生物胁迫反应。     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

Loss of Core 1-derived O-Glycans Decreases Breast Cancer Development in Mice

Mucin-type core 1-derived O-glycans, one of the major types of O-glycans, are highly expressed in mammary gland epithelium. Abnormal O-glycans such as Tn antigen are found in over 90% of breast cancers; however, the in vivo role of these aberrant O-glycans in the etiology of breast cancer is unclear. We generated mice with mammary epithelial specific deletion of core 1-derived O-glycans. By crossing with two spontaneous mouse breast cancer models, we determined that loss of core 1-derived O-glycans delays the onset and progression of breast cancer development. Deficiency of core 1 O-glycosylation impaired the localization of Muc1, a major O-glycoprotein, on the apical surfaces of mammary epithelium. Signaling mediated by Muc1, which is critical for breast cancer development, was also defective in the absence of core 1 O-glycans. This study reveals an unexpected role of core 1-derived O-glycans in breast cancer development in mice.     

Peroxisome Proliferator-activated Receptor-D (PPARD) Coordinates Mouse Spermatogenesis by Modulating Extracellular Signal-regulated Kinase (ERK)-dependent Signaling

Ppard^−/− mice exhibit smaller litter size compared with Ppard^+/+ mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard^−/− mice compared with Ppard^+/+ mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard^+/+ mice as compared with Ppard^−/− mice, and these were associated with decreased Sertoli cell number in Ppard^+/+ mice. Cyclin D1 and cyclin D2 expression was lower in Ppard^+/+ as compared with Ppard^−/− mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard^+/+ mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.