伤寒沙门菌非编码RNA AsrC表达特性及功能初步研究

非编码RNA在细菌的各种生命活动中都发挥着重要的功能.针对伤寒沙门菌非编码RNA AsrC,对其转录和降解特性及其所具有的功能进行初步研究.首先使用Northern blot和qRT-PCR的方法检测了σ因子rpoE和rpoS对AsrC转录的影响,分析了使用利福平抑制细菌RNA合成的情况下,RNase E和RNase III对AsrC降解的影响.其次使用AsrC缺陷株和高表达菌株分析细菌在酸、氧、高渗应激条件下的生长情况.再者通过全基因组芯片技术,研究AsrC高表达菌株基因表达的变化.进一步研究AsrC在细菌侵袭和巨噬细胞胞内生存力中的作用.结果显示,rpoE在酸应激下、rpoS在高渗应激下调节AsrC的转录.细菌中RNase E主要参与了AsrC的降解.高表达AsrC后,有40个基因表达上调,23个基因表达下调.AsrC高表达可以增强伤寒沙门菌对于上皮细胞的侵袭力,并且可以减弱细菌的胞内生存和增殖力.     

Phagosome acidification blocked by intracellular Toxoplasma gondii

Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.     

Trans-spliced leader RNA exists as small nuclear ribonucleoprotein particles in Caenorhabditis elegans

Maturation of some messenger RNAs in the nematode Caenorhabditis elegans involves the acquisition of a 22-base leader at their 5' ends. This 22-base leader, called the spliced leader (SL), is derived from the 5' end of a precursor RNA of 90-100 bases, called spliced leader RNA (SL RNA). SL RNA is transcribed from a 1-kilobase DNA repeat which also encodes the 5S ribosomal RNA. A subset of mRNAs in C. elegans acquire SL from SL RNA by a trans-splicing mechanism. SL behaves as a 5' exon in the trans-splicing reaction. Using antisera against the Sm antigen that is associated with small nuclear ribonucleoprotein particles (snRNPs), we precipitated SL RNA from extracts of C. elegans, indicating that it is bound by the Sm antigen in vivo. SL RNA also possesses the unique trimethylguanosine (m32,2,7G) cap characteristic of most small nuclear RNAs. Therefore, SL RNA is a chimaeric molecule, made up of an snRNA attached to a 5' exon and is a constituent of a snRNP.     

胞外ATP对VK2/E6E7细胞胞内pH的影响

 利用激光扫描共聚焦显微镜（Confocal microscope）采用pH依赖的荧光探针Snarf-4F测量细胞胞内pH(pHi)方法,发现胞外ATP能剂量依赖（10~1 000 μmol/L）地降低人阴道上皮细胞株VK2/E6E7细胞pHi。当胞外加入200 μmol/L ATP时,能快速地使VK2/E6E7细胞pHi降低,此降低的pHi在洗脱ATP后可迅速恢复。这些结果表明胞外ATP能引起VK2/E6E7细胞胞内酸化。     

Cryopyrin activates the inflammasome in response to toxins and ATP

A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.     

Mitochondrial function in normal and diabetic beta-cells.

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic beta-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on beta-cell function reflects the importance of mitochondria in the control of insulin secretion. The beta-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger.