Role of MAPK/ERK in Neurotrophin-4 Potentiation of Necrotic Neuronal Death

Neurotrophic factors have been proposed for the treatment of a variety of neurological diseases. However, to this point they have failed in clinical trials. One potential problem is that while neurotrophic factors attenuate apoptosis, they have the potential to enhance necrosis. In this study we show that neurotrophin-4 (NT-4) attenuated apoptotic neuronal death while potentiating necrotic neuronal death in cortical cultures. The protective effects of NT-4 were not blocked by the mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 or U0126, while the injury potentiation by NT-4 was blocked by these inhibitors. NT-4 stimulated the phosphorylation of ERK1/2 and this phosphorylation was attenuated by U0126 and PD098059. The results indicate a disassociation between the pathway by which NT-4 potentiates necrosis, and that by which it attenuates apoptosis, and suggest that addition of a MEK inhibitor may enhance the beneficial effects of NT-4 in treating complex injuries such as occur in vivo.     

A novel method to analyze leukocyte rolling behavior <Emphasis Type="Italic">in vivo</Emphasis>

Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions. Published: August 27, 2004.     

Mitochondrial permeability transition: a common pathway to necrosis and apoptosis

Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these stresses. In contrast to necrotic cell death which is a consequence of ATP depletion, ATP is required for the development of apoptosis. Reperfusion and the return of normal pH after ischemia initiate the MPT, but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death. Thus, the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Necrosis: a specific form of programmed cell death?

For a long time necrosis was considered as an alternative to programmed cell death, apoptosis. Indeed, necrosis has distinct morphological features and it is accompanied by rapid permeabilization of plasma membrane. However, recent data indicate that, in contrast to necrosis caused by very extreme conditions, there are many examples when this form of cell death may be a normal physiological and regulated (programmed) event. Various stimuli (e.g., cytokines, ischemia, heat, irradiation, pathogens) can cause both apoptosis and necrosis in the same cell population. Furthermore, signaling pathways, such as death receptors, kinase cascades, and mitochondria, participate in both processes, and by modulating these pathways, it is possible to switch between apoptosis and necrosis. Moreover, antiapoptotic mechanisms (e.g., Bcl-2/Bcl-x proteins, heat shock proteins) are equally effective in protection against apoptosis and necrosis. Therefore, necrosis, along with apoptosis, appears to be a specific form of execution phase of programmed cell death, and there are several examples of necrosis during embryogenesis, a normal tissue renewal, and immune response. However, the consequences of necrotic and apoptotic cell death for a whole organism are quite different. In the case of necrosis, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke inflammatory response; during apoptosis these products are safely isolated by membranes and then are consumed by macrophages. The inflammatory response caused by necrosis, however, may have obvious adaptive significance (i.e., emergence of a strong immune response) under some pathological conditions (such as cancer and infection). On the other hand, disturbance of a fine balance between necrosis and apoptosis may be a key element in development of some diseases.     

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells.The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.     

Localization of calcium in the pericarp cells of tomato fruits during the development of blossom-end rot

Summary. Blossom-end rot (BER) of tomato (Lycopersicon esculentum) fruits is considered to be a physiological disorder caused by calcium deficiency. We attempted to clarify the localization of calcium in the pericarp cells and the ultrastructural changes during the development of BER. Calcium precipitates were observed as electron-dense deposits by an antimonate precipitation method. Some calcium precipitates were localized in the cytosol, nucleus, plastids, and vacuoles at an early developmental stage of normal fruits. Calcium precipitates were increased markedly on the plasma membrane during the rapid-fruit-growth stage compared with their level at the early stage. Cell collapse occurred in the water-soaked region at the rapid-fruit-growth stage in BER fruits. There were no visible calcium precipitates on the traces of plasma membrane near the cell wall of the collapsed cells. The amount of calcium precipitates on plasma membranes near collapsed cells was smaller than that in the cells of normal fruits and normal parts of BER fruits, and the amount on cells near collapsed cells was small. The amount of calcium precipitates on the plasma membranes increased as the distance from collapsed cells increased. On the other hand, calcium precipitates were visible normally in the cytosol, organelles, and vacuoles and even traces of them in collapsed cells. The distribution pattern of the calcium precipitates on the plasma membrane was thus considerably different between normal and BER fruits. On the basis of these observations, we concluded that calcium deficiency in plasma membranes caused cell collapses in BER tomato fruits.Correspondence and reprints: National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Ano, Mie, 514-2392, Japan.     

醒脑静注射液联合血府逐瘀汤对SAH大鼠CRP、TNF-alpha的影响

目的：探讨醒脑静注射液联合血府逐瘀汤对蛛网膜下腔出血（subarachnoidhemorrhage,SAH）大鼠C反应蛋白(C-reactionprotein, CRP) 、肿瘤坏死因子-a（Tumor necrosis factor-a,TNF-a）的影响。方法：50 只大鼠随机分为5 组：正常组,模型组,醒脑静组 （0.3 mL/kg）,血府逐瘀汤组（0.3 mL/kg）,联合用药组（醒脑静联合血府逐瘀汤）。除正常组外,其余4组采用" 二次枕大池注血法" 建立大鼠SAH 模型。从造模成功后的第1 d 开始给药,连续给药7 d。正常对照组及模型组给予等量生理盐水。分别在术前、术后 1 d、3 d、7 d、14 d取血浆及脑脊液,按ELISA 试剂盒说明书分别测定各组血清及脑脊液中肿瘤坏死因子-alpha（TNF-alpha）、C- 反应蛋白 （CRP）的含量。结果：（1）术后随着时间推移,SAH大鼠血清及脑脊液中TNF-alpha及CRP 含量逐渐上升,在第7 d 达到高峰。（2）术 后第1 d,与正常组比较,模型组中血清及脑脊液中TNF-琢、CRP含量均显著上升,差异显著（P<0.01）;与模型组比较,醒脑静组及 血府逐瘀汤组中血清及脑脊液中TNF-alpha、CRP 含量无差异（P>0.05）,而联合用药组中血清及脑脊液中TNF-琢含量下降,差异具有 显著意义（P<0.05）。（3）术后第3 d、7 d、14 d,与正常组比较,模型组中血清及脑脊液中TNF-琢、CRP 含量均显著上升,差异显著 （P<0.01）;与模型组比较,醒脑静组、血府逐瘀汤组及联合用药组中血清及脑脊液中TNF-琢、CRP 含量下降,差异均显著（P<0.0 1）,与醒脑静组或血府逐瘀汤组比较,联合用药组中血清及脑脊液中TNF-alpha、CRP含量下降,差异均显著（P<0.05）。结论：醒脑静 联合血府逐瘀汤能有效缓解SAH大鼠CVS,与降低SAH大鼠血清及脑脊中TNF-alpha、CRP 水平有关。     

The serine threonine kinase RIP3: lost and found

Receptor-interacting protein kinase-3 (RIP3, or RIPK3) is an essential protein in the “programmed”, or “regulated” necrosis cell death pathway that is activated in response to death receptor ligands and other types of cellular stress. Programmed necrotic cell death is distinguished from its apoptotic counterpart in that it is not characterized by the activation of caspases; unlike apoptosis, programmed necrosis results in plasma membrane rupture, thus spilling the contents of the cell and triggering the activation of the immune system and inflammation. Here we discuss findings, including our own recent data, which show that RIP3 protein expression is absent in many cancer cell lines. The recent data suggests that the lack of RIP3 expression in a majority of these deficient cell lines is due to methylation-dependent silencing, which limits the responses of these cells to pro-necrotic stimuli. Importantly, RIP3 expression may be restored in many cancer cells through the use of hypomethylating agents, such as decitabine. The potential implications of loss of RIP3 expression in cancer are explored, along with possible consequences for chemotherapeutic response. [BMB Reports 2015; 48(6): 303-312]