白术水提物对酵母朊病毒[PSI^＋]治愈作用的定量研究

目的定量研究白术水提物对酵母朊病毒[PSI^＋]的治愈作用。方法首先用含白术水提物的培养基培养酵母朊病毒[PSI^＋]阳性菌株,初步评价白术的治愈作用。然后在细胞水平借助影印培养法和蛋白水平上使用半变性琼脂糖凝胶电泳结合蛋白免疫技术进一步验证白术水提物对酵母朊病毒[PSI^＋]的治愈作用。结果白术水提物作用酵母朊病毒[PSI^＋]后的治愈率为6％。结论白术水提物对酵母朊病毒[PSI^＋]有一定的治愈作用。     

CRP及PSI评分在社区获得性肺炎中的应用价值

目的探讨C-反应蛋白（CRP）及PSI评分在社区获得性肺炎（CAP）中应用的价值。方法分析我院2009年1月至2010年6月住院CAP患者80例,行PSI评分并进行危险度分级,及重症CAP分组,测定血CRP、白细胞（WBC）。结果 CRP与PSI评分呈显著正相关;重症CAP组PSI、CRP、白细胞均高于非重症组,存在统计学意义;PSI评分不同分级之间：高度组与中度及低度组相比较,CRP有统计学意义,白细胞总数无统计学意义;CRP敏感性高于白细胞。结论 CRP在肺炎诊断中存在重要价值,且可作为重症与非重症肺炎分级的重要指标,及PSI评分系统的一个补充;中、高度PSI评分可作为重症CAP参考指标。     

N-末端脑钠肽前体对社区获得性肺炎严重程度和预后的预测价值

目的研究N-末端脑钠肽前体(NT-proBNP)浓度对社区获得性肺炎(CAP)的严重程度评估和结果预测。方法对200名CAP患者评估临床变量和肺炎严重指数(PSI),同时检测患者的白细胞计数、中性粒细胞计数、C反应蛋白(CRP)、血沉和NT-proBNP,按照PSI评分分组,比较各组NT-proBNP浓度和CRP、血沉、中性粒细胞计数及白细胞计数,评价这些指标与CAP严重程度及预后关系。结果各组NT-proBNP浓度和CRP、血沉、中性粒细胞计数及白细胞计数随着CAP严重程度升高而升高(P<0.01)。死亡患者中NT-proBNP浓度与存活者相比明显升高,差异有统计学意义(P<0.01),与PSI评分组合,NT-proBNP浓度随着病情严重程度的升高而升高,差异明显高于白细胞计数、CRP、血沉及中性粒细胞计数。结论 NT-proBNP可作为CAP患者严重程度分层和预测结果有效的标志物。     

无血清处理对甲状腺癌TT细胞内S100A13及FGF-1释放机制的初步研究

目的 探讨无血清处理人甲状腺髓样癌细胞(TT细胞)内S100A13及成纤维细胞生长因子1( FGF-1)蛋白释放的机制,初步阐明Ca2+在S100A13及FGF-1蛋白释放过程中的作用.方法 Western印迹检测无血清处理TT细胞S100A13及FGF-1蛋白表达;ELISA检测培养上清液FGF-1蛋白浓度;激光共聚焦显微镜实时动态检测无血清处理TT细胞1h内Ca2+浓度([Ca2+]i)变化;间接免疫荧光观察TT细胞内S100A13及FGF-1蛋白荧光分布.结果 无血清处理TT细胞4h和6h后S100A13及FGF-1蛋白表达降低(P＜0.05或P＜0.0l),而TT细胞培养上清液中FGF-1蛋白浓度升高(P＜0.05或P＜0.01).激光共聚焦Ca2+荧光显像发现无血清处理TT细胞23 min内[Ca2+]i保持相对稳定,23 min后[Ca2+]i迅速上升达到峰值1.6μmol/L,第40 win后下降至一个较低水平,第40 min至第60 min[ Ca2+]i接近0.3～0.6 μmol/L.1h内TT细胞平均[Ca2+]i明显高于正常培养组、EGTA组和BAPTA-AM组.加入钙离子螯合剂EGTA组和BAPTA-AM组TT细胞内的S100A13、FGF-1蛋白表达无明显下降.结论 无血清处理诱导TT细胞内S100A13及FGF-1蛋白的释放与[Ca2+]i变化有关;Ca2+螯合剂EGTA、BAPTA-AM能有效拮抗无血清处理引起的TT细胞[Ca2+]i升高,并抑制S100A13及FGF-1蛋白的释放.     

社区获得性肺炎患者的预后与红细胞分布宽度的相关性

目的探讨社区获得性肺炎(CAP)患者的红细胞分布宽度(RDW)与其严重程度及预后的相关性。方法连续性选择2011年7月至2013年7月间,收住上海市嘉定区中心医院急诊科的CAP患者260例,根据RDW是否高于参考值的正常上限分为两组,分别为正常组RDW≦14.5%(n=208)和异常组RDW14.5%(n=52),比较两组患者的临床资料、实验值指标、PSI(pneumonia severity index,肺炎严重程度)评分分级及病死率的差异性;再根据PSI评分及预后,比较不同PSI级别和不同预后的RDW异常率的差异;采用Spearman相关分析对RDW及PSI评分进行相关分析。结果 RDW异常组发生重症率(46.2%vs 24.5%,P=0.003)、PSI IV-V级(53.8%vs 22.1%,P0.001)及病死率(15.4%vs 4.3%,P=0.009)显著高于正常组;RDW异常率随着PSI评分级别增高而增高(9.67%vs 16.13%vs 36.23%vs 60%,P0.001);死亡组发生RDW异常率(18.11%vs 47.06%,P=0.009)显著高于存活组;PSI评分与RDW呈显著正相关(r=0.210,P=0.001)。结论 CAP患者的RDW水平与其严重程度密切相关,RDW14.5%对其预后有重要的参考价值。     

阿魏酸抑制朊病毒复制作用的研究

目的本试验通过已建立的ScN2a细胞模型,探讨阿魏酸对朊病毒复制的影响。方法通过Western blot法检测阿魏酸对朊病毒复制作用的影响。结果0．5、1和5μg／mL的阿魏酸能够极显著的抑制PrPsc的表达水平（P〈0.01）。结论由于中药单体阿魏酸可以抑制ScN2a中PrPsc蛋白的复制,所以中药单体阿魏酸对朊病毒病可能起到一定的治疗作用,这为我们以后对于研究朊病毒的治疗提供了新的思路。     

老年社区获得性肺炎严重度指数PSI和CURB-65评分应用价值研究

目的 分析老年社区获得性肺炎严重度指数PSI和CURB-65评分应用价值;方法取本院住院的老年社区获得性肺炎患者86例作为研究对象,所有患者均符合诊断标准,根据治疗效果不同将所有患者分为有效组与无效组,比较两组患者的疾病情况、PSI及CURB-65评分、敏感性及特异性;结果有效组患者的年龄、合并多种慢性基础疾病、住院时间均明显低于无效组患者（P〈0.05）;治疗4 d后无效组PSI及CURB-65评分明显升高,有效组明显降低,两组间比较差异有显著性（P〈0.05）;PSI评分的敏感性明显高于CURB-65评分（P〈0.05）,特异性低于CURB-65评分（P〈0.05）;结论 PSI和CURB-65评分可以有效判断老年社区获得性肺炎患者的病情严重程度并指导治疗.     

缬沙坦对血管紧张素Ⅱ诱导的肥大心肌细胞胞内钙的影响

目的:观察血管紧张素Ⅱ(AngⅡ)1型受体(AT1)拮抗剂缬沙坦(Valsartan)对AngⅡ诱导的肥大心肌细胞胞内钙([Ca2+]i)变化的影响,并探讨其机制。方法:原代培养的大鼠心肌细胞,3H-亮氨酸掺入法测定心肌细胞蛋白质合成速率,利用共聚焦显微镜技术测定[Ca2+]i。结果:AngⅡ诱导心肌细胞肥大48h后[Ca2+]i明显增加,与空白对照组比较差异有统计学意义(P<0.01);缬沙坦预处理后,[Ca2+]i与肥大组相比下降,差异有统计学意义(P<0.01),与空白对照组相比,差异无统计学意义(P>0.05)。结论:AngⅡ诱导肥大心肌细胞[Ca2+]i增加,缬沙坦通过阻断AT1受体,抑制肥大心肌细胞[Ca2+]i增加。     

槲皮素对家兔主动脉血管平滑肌细胞胞内游离钙浓度的影响

目的: 探讨槲皮素(Que)对血管平滑肌细胞胞内游离钙浓度([Ca2+]i)的影响.方法:采用新一代钙荧光探剂Fluo-3/AM检测Que对培养的兔主动脉血管平滑肌细胞(ASMC) [Ca2+]i 在高K+、去甲肾上腺素(NE)、血管紧张素Ⅱ(AngⅡ)刺激作用下的改变,并与Verapamil进行对照研究.结果:Que(10-6、 10-5、10-4mol/l)呈剂量依赖性抑制高K+去极化引起的[Ca2+]i 升高,与Verapamil作用相似,但弱于Verapa mil;Que (10-6～10- 4mol/L)对NE,AngⅡ通过受体介导引起的[Ca2+]i 增高也具有明显的抑制作用.但Que对静息状态的ASM C[Ca2+]i 无明显影响.结论:Que通过对血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道双重抑制作用,降低细胞内游离钙水平,这可能是其舒血管降压机制之一.     

地塞米松快速抑制气道平滑肌内游离钙升高实验初探

目的 通过对平滑肌细胞行地塞米松快速预处理,拟证实糖皮质激素对平滑肌细胞内游离钙离子浓度[Ca2+]i升高有快速抑制作用.方法 原代培养的大鼠平滑肌细胞,比较不同浓度地塞米松预处理后10 min与对照组之间游离钙上升情况的区别.利用Fura-2/AM显微荧光检测技术,检测肌细胞内[Ca2+]i在受到激动剂刺激后的浓度变化;同时设立RU486及CHX对照组,排除基因组作用在该反应中的影响.结果 地塞米松温浴10 min,能够明显降低Ach引起的气道平滑肌细胞内[Ca2+]i峰值.以上反应均不能被RU486和CHX影响或者逆转.结果 地塞米松能够通过非基因组作用快速抑制Ach引起的气道平滑肌细胞内[Ca2+]i浓度升高.     

Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division

Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers.     

Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride

Guanidine hydrochloride (Gdn.HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn.HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al.[Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Natl Acad Sci USA 102:12789-12794] claimed to show that Gdn.HCl can eliminate the [PSI+] prion from alpha-factor-arrested cells leading them to propose that in Gdn.HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of alpha-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn.HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.     

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI(+)] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI(+)] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI(+)] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are "cured" of [PSI(+)] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI(+)] determinant is not derived from the direct dissolution of self-replicating [PSI(+)] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI(+)] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI(+)] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.     

Amino acid residue 184 of yeast Hsp104 chaperone is critical for prion-curing by guanidine,prion propagation,and thermotolerance

Inactivation of Hsp104 by guanidine is contended to be the mechanism by which guanidine cures yeast prions. We now find an Hsp104 mutation (D184N) that confers resistance to guanidine-curing of the yeast [PSI(+)] prion. In an independent screen we isolated an HSP104 allele altered in the same residue (D184Y) that dramatically impairs [PSI(+)] propagation in a temperature-dependent manner. Directed mutagenesis of HSP104 produced additional alleles that conferred varying degrees of resistance to guanidine-curing or impaired [PSI(+)] propagation. The mutations similarly affected propagation of the [URE3] prion. Basal and induced abundance of all mutant proteins was normal. Thermotolerance of cells expressing mutant proteins was variably resistant to guanidine, and the degree of thermotolerance did not correlate with [PSI(+)] stability. We thus show that guanidine cures yeast prions by inactivating Hsp104 and identify a highly conserved Hsp104 residue that is critical for yeast prion propagation. Our data suggest that Hsp104 activity can be reduced substantially without affecting [PSI(+)] stability, and that Hsp104 interacts differently with prion aggregates than with aggregates of thermally denatured protein.     

Yeast prions [URE3] and [PSI+] are diseases

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises de novo in more than one in 10(6) yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild Saccharomyces strains, we found the [PIN+] prion in 11 strains, but the [URE3] and [PSI+] prions were uniformly absent. In contrast, the "selfish" 2mu DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [PSI+] in wild strains indicates that each prion has a net deleterious effect on its host.     

Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro

Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ+], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ+] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, alpha-synuclein, and synphilin do not. Furthermore, transthyretin and alpha-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states.     

Sex,prions, and plasmids in yeast

Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in ∼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-μm DNA plasmid (∼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (∼23–46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (∼46%). These results show that the detriment of carrying even the mildest forms of [PSI+], [URE3], or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate [PIN+] from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying [PIN+] are far more likely to be heterozygous at RNQ1 and other loci than are [pin−] strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the [PIN+] prion.     

Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion [PSI+

The yeast prion [PSI(+)] provides an epigenetic mechanism for the inheritance of new phenotypes through self-perpetuating changes in protein conformation. [PSI(+)] is a nonfunctional, ordered aggregate of the translation termination factor Sup35p that influences new Sup35 proteins to adopt the same state. The N-terminal region of Sup35p plays a central role in prion induction and propagation. The C-terminal region provides translation termination activity. The function of the highly charged, conformationally flexible middle region (M) is unknown. An M deletion mutant was capable of existing in either the prion or the nonprion state, but in either case it was mostly insoluble. Substituting a charged synthetic polypeptide for M restored solubility, but the prions formed by this variant were mitotically very unstable. Substituting charged flexible regions from two other proteins for M created variants that acquired prion states (defined as self-perpetuating changes in function transferred to them from wild-type [PSI(+)] elements), but had profoundly different properties. One was soluble in both the prion and the nonprion form, mitotically stable but meiotically unstable, and cured by guanidine HCl but not by alterations in heat shock protein 104 (Hsp104p). The other could only maintain the prion state in the presence of wild-type protein, producing Mendelian segregation patterns. The unique character of these M variants, all carrying the same N-terminal prion-determining region, demonstrate the importance of M for [PSI(+)] and suggest that a much wider range of epigenetic phenomena might be based on self-perpetuating, prion-like changes in protein conformation than suggested by our current methods for defining prion states.     

Primary sequence independence for prion formation

Many proteins can adopt self-propagating beta-sheet-rich structures, termed amyloid fibrils. The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are infectious amyloid forms of the proteins Ure2p and Sup35p, respectively. Ure2p forms prions primarily as a result of its sequence composition, as versions of Ure2p with the prion domain amino acids shuffled are still able to form prions. Here we show that prion induction by both Ure2p and Ure2-21p, one of the scrambled versions of Ure2p, is clearly dependent on the length of the inducing fragment. For Ure2-21p, no single sequence is found in all of the inducing fragments, highlighting the sequence independence of prion formation. Furthermore, the sequence of the Sup35p prion domain can also be randomized without blocking prion formation. Indeed, a single shuffled sequence could give rise to several prion variants. These results suggest that [PSI+] formation is driven primarily by the amino acid composition of the Sup35p prion domain, and that the Sup35p oligopeptide repeats are not required for prion maintenance.