"Stem cell" origin of the hematopoietic defect in dyskeratosis congenita

We have used the long-term bone marrow culture (LTBMC) system to analyze hematopoiesis in three patients with dyskeratosis congenita (DC), two of whom had aplastic anemia, and the third had a normal blood count (apart from mild macrocytosis) and normal BM cellularity. Hematopoiesis was severely defective in all three patients, as measured by a low incidence of colony-forming cells and a low level of hematopoiesis in LTBMC. The function of the marrow stroma was normal in its ability to support the growth of hematopoietic progenitors from normal marrows seeded onto them in all three cases, but the generation of hematopoietic progenitors from patients marrow cells inoculated onto normal stromas was reduced, thus suggesting the defect to be of stem cell origin. The parents and unaffected brother of one of the families have also been studied in LTBMC and all showed normal hematopoietic and stromal cell function. From this study we speculate that there are some similarities between DC and the defect in the W/Wv mouse.     

Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5

Hyperphosphorylation of microtubule-associated proteins such as tau and neurofilament may underlie the cytoskeletal abnormalities and neuronal death seen in several neurodegenerative diseases including Alzheimer's disease. One potential mechanism of microtubule-associated protein hyperphosphorylation is augmented activity of protein kinases known to associate with microtubules, such as cdk5 or GSK3beta. Here we show that tau and neurofilament are hyperphosphorylated in transgenic mice that overexpress human p25, an activator of cdk5. The p25 transgenic mice display silver-positive neurons using the Bielschowsky stain. Disturbances in neuronal cytoskeletal organization are apparent at the ultrastructural level. These changes are localized predominantly to the amygdala, thalamus/hypothalamus, and cortex. The p25 transgenic mice display increased spontaneous locomotor activity and differences from control in the elevated plus-maze test. The overexpression of an activator of cdk5 in transgenic mice results in increased cdk5 activity that is sufficient to produce hyperphosphorylation of tau and neurofilament as well as cytoskeletal disruptions reminiscent of Alzheimer's disease and other neurodegenerative diseases.     

Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.     

周期性压力作用下椎间盘细胞中整合素α3的表达

目的 探讨整合素在体外培养的椎间盘细胞力学传导中的作用。方法采用猪的椎间盘进行体外细胞的分离和培养，对细胞施加周期性液压负荷，通过对细胞的形态学观察、Western免疫印迹、免疫细胞化学染色和免疫荧光，分别检测整合素α3和肌动蛋白在周期性压力下椎间盘细胞中的表达。结果经加压后，纤维环细胞和髓核细胞均可见体积缩小，由多角形转变为细长形，α3和肌动蛋白在AF和NP细胞中的表达均明显减少。结论压力抑制了整合素α3的产生，整合素α3将力的信号转换到细胞内时，进一步影响到与其关系密切的肌动蛋白。     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.