Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen‐induced cell killing is still controversial. Our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen‐induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling, and caspase‐3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphate–generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion , after acetaminophen a CsA‐sensitive MPT occurred after 3 to 6 hours followed by a CsA‐insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion–dependent necrosis or caspase‐dependent apoptosis as determined, in part, by ATP availability from glycolysis. (H EPATOLOGY 2004;40:1170–1179.)
The fuel sensing enzyme AMP-activated protein kinase (AMPK) enhances processes that generate ATP when stresses such as exercise or glucose deprivation make cells energy deficient. We report here a novel role of AMPK, to prevent the activation of NF-κB in endothelial cells exposed to the fatty acid palmitate or the cytokine TNF-α. Incubation of cultured human umbilical vein endothelial cells (HUVEC) with elevated levels of palmitate (0.4 mM) increased NF-κB reporter gene expression by 2- to 4-fold within 8 h and caused a 7-fold increase in VCAM-1 mRNA expression at 24 h. In contrast, no increase in reporter gene expression was detected for AP-1, glucocorticoid-, cyclic AMP-, or serum response elements. Similar increases in NF-κB activation and VCAM-1 expression were not observed in cells incubated with an elevated concentration of glucose (25 mM). The increases in NF-κB activation and VCAM-1 expression caused by palmitate were markedly inhibited by co-incubation with the AMPK activator AICAR and, where studied, by expression of a constitutively active AMPK. Likewise, AMPK activation inhibited the increase in NF-κB reporter gene expression observed in HUVEC incubated with TNF-α. The results suggest that AMPK inhibits the activation of NF-κB caused by both palmitate and TNF-α. The mechanism responsible for this action, as well as its relevance to the reported anti-atherogenic actions of exercise, metformin, thiazolidinediones, and adiponectin, all of which have been shown to activate AMPK, remains to be determined.
The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation-contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.
We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
Extracellular action potentials were recorded from developing dissociated rat neocortical networks continuously for up to 49 days in vitro using planar multielectrode arrays. Spontaneous neuronal activity emerged toward the end of the first week in vitro and from then on exhibited periods of elevated firing rates, lasting for a few days up to weeks, which were largely uncorrelated among different recording sites. On a time scale of seconds to minutes, network activity typically displayed an ongoing repetition of distinctive firing patterns, including short episodes of synchronous firing at many sites ( network bursts). Network bursts were highly variable in their individual spatio-temporal firing patterns but showed a remarkably stable underlying probabilistic structure (obtained by summing consecutive bursts) on a time scale of hours. On still longer time scales, network bursts evolved gradually, with a significant broadening (to about 2 s) in the third week in vitro, followed by a drastic shortening after about one month in vitro. Bursts at this age were characterized by highly synchronized onsets reaching peak firing levels within less than ca. 60 ms. This pattern persisted for the rest of the culture period. Throughout the recording period, active sites showed highly persistent temporal relationships within network bursts. These longitudinal recordings of network firing have, thus, brought to light a reproducible pattern of complex changes in spontaneous firing dynamics of bursts during the development of isolated cortical neurons into synaptically interconnected networks.
The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.
Flavones have been classified as anti-atherogenic agents that inhibit monocyte adhesion to stimulated endothelium, possibly by blocking induction of cell adhesion molecules (CAM). This anti-atherogenic feature of these flavonoids appears to be related to their chemical structures. Flavones may interfere with key signaling events involved in endothelial cell activation by inflammatory mediators. This study examined the effects of flavones on the induction of CAM and the translocation and DNA binding of nuclear factor-kappaB (NF-kappaB) in TNF-alpha-activated human umbilical vein endothelial cells (HUVEC). The effects of flavones, luteolin and apigenin, on adhesion of THP-1 monocytes to the TNF-alpha-activated HUVEC, protein expression and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin, and nuclear appearance and DNA binding activity of NF-kappaB were determined. Flavanols, flavonols, and flavanones were used for comparison. TNF-alpha significantly induced HUVEC protein expression of VCAM-1, ICAM-1, and E-selectin with increasing mRNA levels. Luteolin and apigenin inhibited the TNF-alpha-induced upregulation of THP-1 adhesion and VCAM-1 expression; these inhibitory effects were dose-dependent. The flavones at doses of greater than or equal to25 mumol/L almost completely abolished the increased CAM protein and mRNA regardless of their anti-oxidative activity. With the exception of the flavonol quercetin, flavonoids had no such effect; quercetin substantially attenuated the CAM induction. The flavones inhibited nuclear translocation and DNA binding activity of the NF-kappaB-containing binding site in the promoter region of the CAM genes in TNF-alpha-activated HUVEC. The inhibition of endothelial CAM induction by flavones is mediated by their interference with the NF-kappaB-dependent transcription pathway. Thus, the flavones may hamper initial atherosclerotic events involving endothelial CAM induction.
Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of similar to25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.