Role of unphosphorylated transcription factor STAT3 in late cerebral ischemia after subarachnoid hemorrhage.

Molecular mechanisms behind increased cerebral vasospasm and local inflammation in late cerebral ischemia after subarachnoid hemorrhage (SAH) are poorly elucidated. Using system biology tools and experimental SAH models, we have identified signal transducer and activator of transcription 3 (STAT3) transcription factor as a possible major regulatory molecule. On the basis of the presence of transcription factor binding sequence in the promoters of differentially regulated genes (significant enrichment PE: 6 × 10(5)) and the consistent expression of STAT3 (mRNA, P=0.0159 and Protein, P=0.0467), we hypothesize that unphosphorylated STAT3 may directly DNA bind and probably affect the genes that are involved in inflammation and late cerebral ischemia to influence the pathologic progression of SAH.

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes.

Loss of caspase-9 reveals its essential role for caspase-2 activation and mitochondrial membrane depolarization.

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis.

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.

Viral modulation of cell death by inhibition of caspases.

Caspases are key effectors of the apoptotic process. Some of them play important roles in the immune system, being involved in the proteolytic maturation of the key cytokines, including interleukin 1beta (IL-1beta) and IL-18. The latter directs the production of interferon gamma (IFN-gamma). Among pathogens, particularly viruses express various modulators of caspases that inhibit their activity by direct binding. By evading the apoptotic process, viruses can better control their production in the infected cell and avoid the attack of the immune system. Targeting the maturation of the key cytokines involved in the initiation of (antiviral) immune response helps to avoid recognition and eradication by the immune system. The three main classes of caspase inhibitors frequently found among viruses include serine proteinase inhibitors (serpins: CrmA/SPI-2), viral IAPs (vIAPs) and p35. Their molecular mechanisms of action, structures and overall influence on cellular physiology are discussed in the review below.

The role of caspases in cryoinjury: caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells.

Cryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase-3 is induced during the freeze-thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD-fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony-forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl-2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjury-associated cell death is mediated by a Bcl-2-controlled mitochondrial pathway. Thus, our data suggest the use of zVAD-fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.