The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks repair. Ku is also expressed on the cell surface of different types of cells where its function remains poorly understood. From a yeast two-hybrid screen, we have identified a specific interaction between the core region of Ku80 and the hemopexin domain of metalloproteinase 9 (MMP-9), a key enzyme involved in the degradation of extracellular matrix (ECM) components. Ku associates with MMP-9 on the surface of leukemic cells as demonstrated by co-immunoprecipitation experiments in membrane extracts and double-label immunofluorescence studies. In normal and tumoral migratory cells, Ku80 and MMP-9 colocalize at the periphery of leading edge of cells and cellular invasion of collagen IV matrices was blocked by antibodies directed against Ku70 or Ku80 subunits as well as by Ku80-specific antisense oligonucleotides. Our results indicate that Ku and MMP-9 interact at the cell membrane of highly invasive hematopoietic cells of normal and tumoral origin and document the unexpected importance of the membrane-associated form of Ku in the regulation of ECM remodelling.     

A Dexamethasone Prodrug Reduces the Renal Macrophage Response and Provides Enhanced Resolution of Established Murine Lupus Nephritis

We evaluated the ability of a macromolecular prodrug of dexamethasone (P-Dex) to treat lupus nephritis in (NZB × NZW)F1 mice. We also explored the mechanism underlying the anti-inflammatory effects of this prodrug. P-Dex eliminated albuminuria in most (NZB × NZW)F1 mice. Furthermore, P-Dex reduced the incidence of severe nephritis and extended lifespan in these mice. P-Dex treatment also prevented the development of lupus-associated hypertension and vasculitis. Although P-Dex did not reduce serum levels of anti-dsDNA antibodies or glomerular immune complexes, P-Dex reduced macrophage recruitment to the kidney and attenuated tubulointerstitial injury. In contrast to what was observed with free dexamethasone, P-Dex did not induce any deterioration of bone quality. However, P-Dex did lead to reduced peripheral white blood cell counts and adrenal gland atrophy. These results suggest that P-Dex is more effective and less toxic than free dexamethasone for the treatment of lupus nephritis in (NZB × NZW)F1 mice. Furthermore, the data suggest that P-Dex may treat nephritis by attenuating the renal inflammatory response to immune complexes, leading to decreased immune cell infiltration and diminished renal inflammation and injury.     

PML coiled-coil结构域与RAN结合蛋白9相互作用的验证

目的验证前髓细胞性白血病的卷曲螺旋结构域（PML—C）和RAN结合蛋白9（RANBP9）之间的相互作用。方法构建分别表达诱饵蛋白PML—C和靶蛋白RANBP9的载体pGBKT7-PML-C和pACT2-RANBP9,然后转人酵母AH109,培养3～5d后对其是否有胞内相互作用进行检测。将目的片断PML-C和RANBP9再次构建于真核生物表达载体pCMV—HA和pCMV—myc里,然后共转染人胚肾293细胞里（HEK293）,最后对其是否有体外相互作用通过免疫共沉淀和免疫印迹进行分析。结果在共转化了质粒pGBKT7-PML—C和pACT2-RANBP9的AH109酵母平板里观察到蓝色菌落生长。用抗HA多克隆抗体对共转染过重组质粒的HEK293细胞的蛋白提取物进行免疫共沉淀,再用抗myc单克隆抗体作为一抗进行免疫印迹,最终检测出融合蛋白myc—RANBP9条带。结论酵母双杂交实验验证了PML—C和RANBP9之间存在胞内相互作用,同时免疫共沉淀实验也从体外验证了它们之间的相互作用。     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.     

NaCl胁迫对马齿苋光合作用及叶绿素荧光特性的影响

以马齿苋为材料,采用温室盆栽法研究了14 d NaCl胁迫处理对其幼苗生长、光合作用和叶绿素荧光特性的影响.结果显示:(1)马齿苋幼苗的鲜重和株高在25 mmol·L-1 NaCl胁迫时与对照无显著差异,但其随着NaCl浓度的继续增加均显著降低,且其生物量受到的抑制早于株高.(2) NaCl胁迫下,马齿苋幼苗叶片净光合速率(Pn)降低,胞间二氧化碳浓度(C1)增大,且两者的变化幅度随着NaCl浓度增加而增大.(3)NaCl胁迫下,马齿苋幼苗叶片的初始荧光(F0)、最大荧光(Fm)、可变荧光(Fv)、恒态荧光(Fs)、恒态荧光与初始荧光差值(△F0)、PSⅡ潜在光化学效率(Fv/Fo)和PSⅡ最大光化学效率(Fv/Fm)均降低,叶片光化学荧光猝灭系数(qP)也在NaCl胁迫下降低,而非光化学荧光猝灭系数(NPQ)则上升;在0～50 mmol·L-1NaCl胁迫下,幼苗叶片各荧光参数下降幅度小于其他高浓度NaCl胁迫.研究表明,在NaCl胁迫条件下,马齿苋幼苗叶片的光合作用受光抑制伤害,但在低浓度NaCl下能够较多地将光能用于光化学反应,光抑制程度较低,保持了较高的净光合速率,明显减轻盐胁迫对植株生长的影响,表现出一定的耐盐性.