Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system

A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB.     

Computational fluid dynamics analysis for process impact assessment during thermal pasteurization of intact eggs

Transient temperature and albumen velocity profiles during thermal pasteurization of intact eggs were studied using a commercial computational fluid dynamics (CFD) package. Simulated temperature profiles were in close agreement with experimental data for eggs of different sizes. Convective heat transfer only occurred in the egg white fraction, and conductive heat transfer only occurred in the yolk. For process assessment, a generally accepted kinetic inactivation model for Salmonella Enteritidis was incorporated into the CFD analysis. Minimum process times and temperatures needed to provide equivalent pasteurization at 5-log reductions of the target microorganism were obtained on a theoretical basis. The combination of CFD analysis and inactivation kinetics can be very useful for assessing pasteurization of intact eggs and can enable processors to gain a better understanding of these processes and to establish process conditions for consumer-safe eggs.     

Telecommunications network architecture

An abstract generic system model and methodology that form the Telecommunications Network Architecture (TNA) are described. The model represents controls and communication functions required for the transfer of information within a node or a network of nodes providing any service (message, packet, or voice). The architecture can be used to represent the most primitive switching mechanism. The methodology is intended to offer the system designer an appreciation of communication systems, and the ability to trace the impact of modifications.