Background. Protein Kinase C (PKC) is a family of enzymes that plays a key role in cell signaling pathways leading to cellular activation and proliferation. Conventional PKC (cPKC) is dependent on calcium for activation. We have proposed that cyclosporin A (CsA), despite being a calcineurin inhibitor, will activate PKC in B cells, thus promoting Epstein-Barr virus (EBV)-induced transformation. Here we show that CsA promoted atypical PKC isoform PKC-zeta in B cells.Materials and methods. Western-blot was used to assay PKC-zeta protein level in EBV-B cells. Confocal microscopy was used to assay PKC-zeta translocation from cytosol to cell membrane, a known process of PKC activation.Results. CsA (500 ng/mL) time dependently increased PKC-zeta from control of 7055 units to 7145, 10,805, 10,914, and 12,705 units, respectively, after 15 min, 1 h, 12 h, and 24 h of incubation in EBV-transformed human B-cell line (LCL). CsA increased PKC-zeta expression was inhibited 50% by Vit.E (40 mu M) indicating that this effect may be due to oxidative stress induced by CsA. Indeed, after oxidant H2O2 (0.1 mM) treatment, PKC-zeta protein level in LCL cells increased 124%, 257%, 349%, and 359% after 15 min, 1 h, 12 h, and 24 h of culture compared with control. Addition of Vit.E (40 mu M) in H2O2 (0.1 mM) treatment and then with Vit.E in the culture decreased PKC-zeta level in LCL cells 26%,20%,41%, and 60% after 15 min, 1 h, 12 h, and 24 h of culture. In confocal microscopy in Jurkat T cell line, phorbol 12-myristate 13-acetate (PMA) activated cPKC isoform PKC alpha after 30 min treatment and activated PKC-zeta after 60 min treatment. CsA inhibited PMA activation of PKC-alpha but not PKC-zeta. CsA alone did not activate PKC-alpha or PKC-zeta in Jurkat T cells. In LCL and in EBV-infected human B-cells, PMA stimulated PKC-alpha activation after 30 min treatment and stimulated PKC-zeta activation after 60 min treatment. CsA inhibited PMA activation of PKC-alpha but not PKC-zeta. In addition, CsA activated PKC-zeta in the EBV-transformed and EBV-infected human B cells.Conclusion. These experiments show that CsA-induced oxidative stress caused PKC-zeta up-regulation in LCL cells, and show the differential effect of CsA in the PKC signaling pathways in T cells versus B cells. CsA-induced PKC-zeta activation may be an important signaling step in EBV-induced post-transplant lymphoproliferative disorders. (C) 2009 Elsevier Inc. All rights reserved.

Background: Orderly cell death, termed apoptosis, features a morphology that is distinct from necrotic, or accidental, cell death. As the body of literature on apoptotic cell death grows, it is difficult for practicing surgeons to stay current with the involved mechanisms and their biologic significance. Methods: A MEDLINE/PubMed literature search was conducted, followed by manual crossreferencing, to identify relevant articles published in the English language between 1972 and 2004. Results: Apoptosis is now known to be involved in numerous disease states. Ischemia-reperfusion injury and acute pancreatitis are but two surgical entities in which the balance of apoptotic and necrotic cell death has a profound effect on clinical outcome. Similarly, the timing and extent of apoptosis in immune cells are important factors that determine the outcome of septic patients. Conclusions: As already demonstrated in animal models, further research in this field will target opportunities for therapeutic intervention, making it increasingly important for clinicians to be familiar with apoptosis and necrosis, and their roles in normal and pathologic states.

Lipopolysaccharide (LPS) is known to prolong the functional lifespan of neutrophils at a site of infection by preventing apoptosis through inhibitor of apoptosis proteins (IAPs). We hypothesized that the increased neutrophil lifespan ultimately leads to a larger fraction of cells undergoing uncontrolled, primary necrosis. Diluted venous whole blood was incubated with increasing concentrations of LPS for up to 36 hr. The percentages of apoptotic, necrotic and viable neutrophils were assessed using the Annexin V/propidium iodide flow cytometric assay. LPS led to a reduction of neutrophil apoptosis and increased the number of viable cells at 12, 24, and 36 hr of incubation. At the same time intervals, there was a significant increase in the percentage of cells undergoing primary necrosis for all concentrations of LPS (e.g., 10 ng/ml LPS at 24 h produced a mean increase from 9.6% in controls to 30.6%, p < 0.001). This increase in direct neutrophil necrosis following LPS activation may amplify local proinflammatory effects through less well controlled release of neutrophil contents into surrounding tissue.

This paper describes the integration of Design-ForTestibility (DFT) processes, tools, and methodologies into the RASSP System, which consists of the Design Environment and Enterprise System. The blueprint for the DFT developments is the DFT Methodology, which is highly automated, hierarchical, and spans the entire life cycle, contributing significantly to the RASSP goals of 4x improvement in cycle time, design quality, and life cycle costs. Key concepts of the DFT methodology are covered. A preferred testability architecture that encompasses embedded test resources (BIST), external test resources (ATE), and testbenches for design verification is described. Integration of the DFT methodology and testability architecture into the RASSP system is covered. The paper concludes with a discussion of the contribution of DFT to meeting the RASSP goals.