Design of Digital Window Comparators and their Implementation within Mixed-Signal DfT Schemes

An in detail design of digital window comparators is presented. This comparator can be used for the on-chip (and potentially on-line) response evaluation of analogue circuits. The analysis shows that if the design parameter is in the order of 1 ... 0,36 for the NOR and 1 ... 2,8 for the NAND good results for the comparator can be achieved and the variation of the window position is limited to 5%. Parameter and temperature drifts are discussed along with results from characterisation. The results can be extended to deep-submicron technologies if the respective equations are used to derive the logical threshold and beta values. A simplified comparator is described that also allows the localisation of the evaluated signal. The conditions for the implementation of the window comparators into Design-for-Testability schemes are outlined. It is demonstrated that the digital window comparator can be implemented in the digital part of the mixed-signal integrated circuit.     

<Emphasis Type="Italic">In vivo</Emphasis> measurement of the redox state

As part of an aerobic life, we oxidize a large pool of biomolecules to obtain chemical energy. During this process, several intermediates are formed; some are chemically unstable and are referred to as free radicals (FR). FR tend to react quickly with their surrounding biological environment; depending on the nature of the molecule attacked, different reactions can occur, i.e., lipid peroxidation, protein oxidation, or DNA oxidation products. As aerobic life has evolved, antioxidant defense systems against FR have developed. When an imbalance between production of FR (oxidants) and defese systems against them (antioxidants) happens, a situation of oxidative stress occurs. This can lead to irreversible biochemical changes, with subsequent tissue damage and disease. Establishing the involvement of FR in the pathogenesis of a disease has been difficult because of the lack of sensitie and specific methodology to detect them. No ideal biomarkers for in vivo FR-induced damage are available as yet. However, some reliable indices of FR formation are now available, and in some pathologic conditions, evidence is accumulating to show that FR damage might play a functional role. The task for the near future will be to try to simplify the analytical methodology and elucidate the molecular mechanisms underlying the formation, disposition, and kinetics of FR marker molecules.     

Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src

Quiescent mammalian fibroblasts can be induced to reenter the cell cycle by growth factors and oncoproteins. We studied the pathway(s) through which v-Src, the oncogenic tyrosine kinase encoded by the v-src oncogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to enter S-phase. To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele from the mutant ts LA31 PR-A. NIH(MR31) cells displayed a temperature-conditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature. Serum stimulation or thermolabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kinases with very little tyrosine phosphorylation of the Shc adaptor proteins at least during the early stages of v-Src reactivation and that v-Src-induced S-phase entry was strongly inhibited by drugs affecting MEK or PI 3-kinase. Our results also suggest that down-regulation of gas1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 down-regulation does not require PI 3-kinase dependent signals.     

Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol

BACKGROUND & AIMS: We reported previously that patients with alcoholic liver disease (ALD) have circulating immunoglobulins reacting with cytochrome P4502E1 (CYP2E1) complexed with hydroxyethyl free radicals. The aim of this study was to investigate whether hydroxyethyl radical adducts are present on the plasma membranes of ethanol-treated hepatocytes and their role in antibody-dependent cytotoxicity. METHODS: Immunofluorescence confocal laser microscopy, Western blotting, and antibody-dependent cell-mediated cytotoxicity assay were used. RESULTS: Isolated rat hepatocytes incubated in vitro with ethanol or obtained from ethanol-treated animals showed strong surface fluorescence when exposed to rabbit anti-hydroxyethyl radical serum or sera from patients with ALD. No surface fluorescence was evident on control hepatocytes or after scavenging hydroxyethyl radicals with 4-pyridyl-1-oxide-t-butyl nitrone. The presence of CYP2E1-hydroxyethyl radical adducts on hepatocyte plasma membranes was shown by Western blot and by immunofluorescence using double staining for human and rabbit anti-CYP2E1 immunoglobulin G. Cytotoxicity was observed in ethanol-treated hepatocytes incubated with immunoglobulin G from patients with ALD and normal human blood mononuclear cells. This effect was blocked by preabsorbing the sera with human albumin complexed with hydroxyethyl radicals, which also eliminated the antibody reaction with the plasma membranes. CONCLUSIONS: Hydroxyethyl radicals bound to CYP2E1 on hepatocyte plasma membranes can target immune reactions triggered by alcohol abuse.     

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.