Microinversions in mammalian evolution.

We propose an approach for identifying microinversions across different species and show that microinversions provide a source of low-homoplasy evolutionary characters. These characters may be used as "certificates" to verify different branches in a phylogenetic tree, turning the challenging problem of phylogeny reconstruction into a relatively simple algorithmic problem. We estimate that there exist hundreds of thousands of microinversions in genomes of mammals from comparative sequencing projects, an untapped source of new phylogenetic characters.

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation.

Tumor necrosis factor (TNF) signaling through TNF receptor 1 (TNFR1) with downstream participation of nuclear factor kappaB (NFkappaB), interleukin 6 (IL-6), and signal transducers and activators of transcription 3 (STAT3) is required for initiation of liver regeneration. It is not known whether the proliferative effect of TNF on hepatocytes is direct or requires the participation of Kupffer cells, the liver resident macrophages. Moreover, it has not been determined whether NFkappaB activation is an essential step in TNF-induced proliferation. To answer these questions, we conducted studies in LE6 cells, a rat liver epithelial cell line with hepatocyte progenitor capacity. We report that TNF induces DNA replication in growth-arrested LE6 cells and that its effect involves the activation of NFkappaB and STAT3 and an increase in c-myc and IL-6 mRNAs. All of these effects, which mimic the events that initiate liver regeneration in vivo, are blocked if NFKB activation is inhibited by expression of a dominant-inhibitor IkappaBalpha mutant (deltaN-IkappaBalpha). Although NFkappaB blockage by deltaN-IkappaBalpha causes caspase activation and massive death of cells stimulated by TNF, inhibition of NFkappaB and STAT3 binding by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone results in G0-G1 cell cycle arrest without death. We conclude that NFkappaB is an essential component of the TNF proliferative pathway and that TNF-induced changes in IL-6 mRNA, STAT3, and c-myc mRNA are dependent on NFkappaB activation. Blockage of NFkappaB inhibits TNF-induced proliferation but does not necessarily cause cell death.

NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival.

The alphavbeta3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-kappaB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and beta3 integrin ligation rapidly increased NF-kappaB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a beta1-integrin ligand) did not induce NF-kappaB activity. The alphavbeta3 integrin was most important for osteopontin-mediated NF-kappaB induction and survival, since adding a neutralizing anti-beta3 integrin antibody blocked NF-kappaB activity and induced endothelial cell death when cells were plated on osteopontin. NF-kappaB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-kappaB activity with nonphosphorylatable IkappaB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-kappaB was not required for fibronectin, laminin, and collagen type I-induced survival. Activation of NF-kappaB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-kappaB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-kappaB activation. These studies identify NF-kappaB as an important signaling molecule in alphavbeta3 integrin-mediated endothelial cell survival.

Ethanol administration alters the proteolytic activity of hepatic lysosomes.

Protein accumulation in liver cells contributes to alcohol-induced hepatomegaly and is the result of an ethanol-elicited deceleration of protein catabolism (Alcohol Clin Exp Res 13:49, 1989). Because lysosomes are active in the degradation of most hepatic proteins, the present studies were conducted to determine whether ethanol administration altered the proteolytic activities of partially purified hepatic lysosomes. Rats were fed liquid diets containing either ethanol (36% of calories) or isocaloric maltodextrin for periods of 2-34 days. Prior to death, all animals were injected with [3H]leucine to label hepatic proteins. Rats subjected to even brief periods of ethanol feeding (2-8 days) exhibited significant hepatomegaly and hepatic protein accumulation compared with pair-fed control animals. Crude liver homogenates and isolated lysosomal-mitochondrial and cytosolic subfractions were incubated at 37 degrees C, and the acid-soluble radioactivity generated during incubation was measured as an index of proteolysis. At neutral pH, in vitro protein breakdown in incubated liver homogenates and subcellular fractions from control and ethanol-fed rats did not differ significantly. The extent of protein hydrolysis increased when samples were incubated at pH 5.5, which approximates the pH optimum for catalysis by lysosomal acid proteases. Under the latter conditions, partially purified lysosomes from control animals had 2-fold higher levels of proteolysis than corresponding fractions from ethanol-fed rats. The difference in proteolytic capacity appeared to be related to a lower latency and a higher degree of fragility of lysosomes from ethanol-fed rats at the acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma protein catabolism in ethanol- and colchicine-treated liver slices.

The present study was conducted to determine whether the antisecretory agents colchicine and ethanol affect the intracellular degradation of plasma proteins in rat liver. Plasma proteins were prelabeled in vivo with [3H]leucine and their levels were monitored immunochemically in both the medium and extracts of rat liver slices incubated alone or in the presence of 50 microM colchicine or 25 mM ethanol. Compared with those left untreated, colchicine-treated slices had a 40-55% lower secretory capacity and, at one point, showed significant hepatocellular retention of total plasma proteins. Plasma protein secretion by ethanol-treated liver slices was 22-32% lower than controls, but there was no detectable retention of unsecreted plasma proteins in the ethanol-treated liver tissue. In all experiments, the total radioactivity in plasma proteins (i.e., the immunoprecipitable radioactivity in the liver plus that in the medium) decreased with time in a manner suggestive of intracellular degradation. Regression analyses of the rates of degradation of presecretory proteins revealed that compared with controls, plasma protein catabolism was accelerated 57% in colchicine-treated slices. In ethanol-treated liver slices, there was a 50% increase in the degradation of total plasma proteins and a 46% increase in albumin catabolism. In all cases, degradation was intracellular. These findings indicate that inhibition of hepatic protein secretion by either colchicine or ethanol is associated with accelerated catabolism of unsecreted plasma proteins, suggesting that hepatocellular degradative processes are responsive to changes in the levels of presecretory proteins and/or perturbations of the secretory process.