Specificity of short interfering RNA determined through gene expression signatures

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.     

Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia

Heterozygous mutations in the CD95 (APO-1/Fas) receptor occur in most individuals with autoimmune lymphoproliferative syndrome (ALPS) and dominantly interfere with apoptosis by an unknown mechanism. We show that local or global alterations in the structure of the cytoplasmic death domain from nine independent ALPS CD95 death-domain mutations result in a failure to bind the FADD/MORT1 signaling protein. Despite heterozygosity for the abnormal allele, lymphocytes from ALPS patients showed markedly decreased FADD association and a loss of caspase recruitment and activation after CD95 crosslinking. These data suggest that intracytoplasmic CD95 mutations in ALPS impair apoptosis chiefly by disrupting death-domain interactions with the signaling protein FADD/MORT1.     

Nanoparticle-mediated Gene Silencing Confers Radioprotection to Salivary Glands In Vivo

Radiation treatment of head and neck cancers causes irreversible damage of the salivary glands (SG). Here, we introduce a preclinical mouse model for small-interfering RNA (siRNA)-based gene silencing to provide protection of SG from radiation-induced apoptosis. Novel, pH-responsive nanoparticles complexed with siRNAs were introduced into mouse submandibular glands (SMG) by retroductal injection to modulate gene expression in vivo. To validate this approach, we first targeted Nkcc1, an ion transporter that is essential for saliva secretion. Nkcc1 siRNA delivery resulted in efficient knockdown, as quantified at the mRNA and the protein levels, and the functional result of Nkcc1 knockdown phenocopied the severe decrease in saliva secretion, characteristic of the systemic Nkcc1 gene knockout. To establish a strategy to prevent apoptotic cell loss due to radiation damage, siRNAs targeting the proapoptotic Pkcδ gene were administered into SMG before ionizing radiation. Knockdown of Pkcδ not only reduced the number of apoptotic cells during the acute phase of radiation damage, but also markedly improved saliva secretion at 3 months in irradiated animals, indicating that this treatment confers protection from hyposalivation. These results demonstrate that nanoparticle delivery of siRNAs targeting a proapoptotic gene is a localized, nonviral, and effective means of conferring radioprotection to the SGs.     

ACE抑制剂卡托普利对实验性围手术期心肌缺血的影响

目的:图手术期心肌缺血主要是因为应激引起冠状动脉内皮功能障碍所致,所以观察卡托普利对其影响。方法:杂种犬20只均分为4组:Ⅰ组(对照组),Ⅱ组(心肌梗塞模型组),Ⅲ组(心梗 胃大部切除术)和Ⅳ组(心梗 卡托普利 胃大部切除术)。心梗2周后行胃大部分切除术,测定Ⅲ、Ⅳ两组的基础状态、术前和术后的血流动力学指标、血浆内皮素(ET)及一氧化氮(NO)。用组织原位杂交方法观察4组非梗塞区冠脉内皮-氧化氮合酶(NOS)mRNA表达水平。结果:在Ⅲ组,手术使LV dP/dt_(max)、心脏指数(CI)及NO下降,引起LVEDP、PCWP、总外周阻力(TPR)、左室舒张压力下降时间常数(T值)和ET升高。在Ⅳ组,用卡托普利后40min,TPR下降,T值升高;手术使血流动力学指标回降,不影响其它指标。组织原位杂交示,NOS mRNA在Ⅰ组高度表达,Ⅱ组和Ⅳ组次之,Ⅲ组最低。结论:卡托普利能预防胃大部切除术引起的左室舒缩障碍和冠脉内皮功能障碍。     

Intraspinal synovial cysts: MR imaging

Juxtaarticular intraspinal synovial cysts are unusual lesions of the spine associated with facet arthropathy. These lesions can cause radicular symptoms and may masquerade clinically as other, more common entities. Synovial cysts have been detected at myelography and have been well characterized at computed tomography as posterolateral epidural masses, typically at L4-5. Six synovial cysts of the lumbar spine were demonstrated on magnetic resonance (MR) images. The signal-intensity patterns of these lesions are variable. MR imaging can be used to document the presence of hemorrhage within the cyst, which may relate to the exacerbation of symptoms. Air-filled synovial cysts may be difficult to detect and distinguish from facet arthropathy.     

Corpus callosum and limbic system: neuroanatomic MR evaluation of developmental anomalies

Agenesis of the corpus callosum is a complex malformation of the brain that has been associated with varying degrees of limbic system maldevelopment. We retrospectively reviewed the records of 11 patients with callosal agenesis (seven total, four partial) who underwent magnetic resonance (MR) imaging, with particular attention to the associated malformations of the limbic system. Comparison was made with selected images from MR examinations of healthy volunteers and with necropsy specimens from other patients with callosal agenesis. Ten of 11 patients demonstrated limbic anomalies (severe motion artifact precluded evaluation of these structures in one patient). MR depicted not only the abnormalities intrinsic to callosal agenesis but also the frequently associated malformations of the limbic system.     

The clinical features of immunoglobulin light-chain (AL) amyloidosis with heart involvement

We reviewed clinical presentation, investigations, therapy, prognosis and outcome of 232 patients with primary (AL) cardiac amyloidosis. There were 142 men and 90 women. Median age at presentation was 59 years (range 29-85). AL heart disease was unusual both in patients under the age of 40 (3.0%) and in non-Caucasians (6.5%). Fatigue and weakness were the commonest presenting symptoms. Hallmark features of periorbital ecchymoses and macroglossia were present in 12.5% and 27.2%, respectively. AL cardiac amyloidosis was unusual in isolation (3.9%), and most frequently patients had features of multiorgan dysfunction; heavy proteinuria and features of malabsorption predominating in this respect. Heart involvement represents the worst prognostic indicator, with a median survival from diagnosis of 1.08 years, falling to 0.75 years with the onset of heart failure. Current therapeutic procedures appear to prolong survival, with left ventricular wall thickness, mass and ejection fraction on echocardiography and late potentials on signal averaged electrocardiography of use in prognostic stratification. Cardiac involvement from AL amyloidosis is rapidly fatal. It should be suspected in all patients with heart failure who have wall thickening on echo, normal chamber sizes, low EKG voltages and evidence suggesting a multisystem disease.