A more serine way to die: defining the characteristics of serine protease-mediated cell death cascades.

The morphological features observed by Kerr, Wylie and Currie in 1972 define apoptosis, necrosis and autophagy. An appreciable number of alternative systems do not fall neatly under these categories, warranting a review of alternative proteolytic machinery and its contribution to cell death. This review aims to pinpoint key molecular features of serine protease-mediated pro-apoptotic signalling. The profile created will contribute to a standard set of biochemical criteria that can serve in differentiating within cell death subtypes.

Endothelial cell response to biomechanical forces under simulated vascular loading conditions.

In vivo, endothelial cells (EC) are constantly exposed to the haemodynamic forces (HF) of pressure, wall shear stress and hoop stress. The main aim of this study was to design, create and validate a novel perfusion bioreactor capable of delivering shear stress and intravascular pressure to EC in vitro and to characterise their morphology, orientation and gene expression. Here we report the creation and validation of such a simulator and the dual application of pressure (120/60 mmHg) and low shear stress (5 dyn/cm(2)) to a monolayer of EC established on a non-compliant silicone tube. Under these conditions, EC elongated and realigned obliquely to the direction of applied shear stress in a time-dependent manner. Furthermore, randomly distributed F-actin microfilaments reorganised into long, dense stress fibres crossing the cells in a direction perpendicular to that of flow. Finally, combinatorial biomechanical conditioning of EC induced the expression of the inflammatory-associated E-selectin gene.

Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis.

Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

Characterization of a serine protease-mediated cell death program activated in human leukemia cells.

Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.

Bioreactors for cardiovascular cell and tissue growth: a review.

Heart disease is a major cause of death in the Western world. In the past three decades there has been a number of improvements in artificial devices and surgical techniques for cardiovascular disease; however, there is still a need for novel devices, especially for those individuals who cannot receive conventional therapy. The major disadvantage of current artificial devices lies in the fact that they cannot grow, remodel, or repair in vivo. Tissue engineering offers the possibility of developing a biological substitute material in vitro with the inherent mechanical, chemical, biological, and morphological properties required in vivo, on an individual patient basis. In order to develop a true biological cardiovascular device a dynamic physiological environment needs to be created. One approach that employs the use of a simulated biological environment is a bioreactor in which the in vivo biomechanical and biochemical conditions are created in vitro for functional tissue development. A review of the current state of the art bioreactors for the generation of tissue engineered cardiovascular devices is presented in this study. The effect of the simulated physiological environment of the bioreactor on tissue development is examined with respect to the materials properties of vascular grafts, heart valves, and cardiac muscles developed in these bioreactors.

A role for HNF-3 in the regulation of the HNF-1 gene of the Atlantic salmon.

Hepatocyte nuclear factors -1 (HNF-1) and -3 (HNF-3) are hepatocyte-enriched transcription factors that are central to the establishment and maintenance of the liver phenotype in vertebrates. In the present study we demonstrate that, in the Atlantic salmon, asHNF-3 regulates the expression of the gene for asHNF-1. Multiple putative binding sites for asHNF-3 were identified within the 5' flanking region of the HNF-1 gene using a computer-based algorithm, and these were confirmed to be functional by electrophoretic mobility shift assays. In transient transfection assays it was shown that co-expression of asHNF-3 leads to a decrease in the promoter activity of the 5' flanking region of the asHNF-1 gene.