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The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1.sup.1. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death.sup.2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype.sup.9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1.sup.-/- (also known as Rbck1.sup.-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3.sup.-/-Casp8.sup.-/-Hoil-1.sup.-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.The HOIL-1 component of the LUBAC ubiquitin ligase complex is required for LUBAC activity, which prevents lethality during embryogenesis by preventing aberrant TNFR1-mediated endothelial cell death and RIPK1-mediated defects in haematopoiesis., Author(s): Nieves Peltzer [sup.1] , Maurice Darding [sup.1] , Antonella Montinaro [sup.1] , Peter Draber [sup.1] [sup.2] , Helena Draberova [sup.1] [sup.2] , Sebastian Kupka [sup.1] , Eva Rieser [sup.1] [...]

Zinc is an essential micronutrient and cytoprotectant involved in the host response to inflammatory stress. We tested whether zinc transporters, the critical regulators that maintain intracellular zinc concentrations, play a role in cell survival, particularly in lung epithelia, during inflammation. Initially, mRNA transcripts were quantitatively measured by RT-PCR for all known human zinc transporters, including 14 importers (SLC39[A.sub.1-14]) and 10 exporters (SLC30[A.sub.1-10]), in primary human lung epithelia obtained from multiple human donors and BEAS-2B cell cultures under baseline and TNF-[alpha]-stimulated conditions. While many zinc transporters were constitutively expressed, only SLC39A8 (Zip8) mRNA was strongly induced by TNF-[alpha]. Endogenous Zip8 protein was not routinely detected under baseline conditions. In sharp contrast, TNF-[alpha] induced the expression of a glycosylated protein that translocated to the plasma membrane and mitochondria. Increased Zip8 expression resulted in an increase in intracellular zinc content and coincided with cell survival in the presence of TNF-[alpha]. Inhibition of Zip8 expression using a short interfering RNA probe reduced cellular zinc content and impaired mitochondrial function in response to TNF-[alpha], resulting in loss of cell viability. These data are the first to characterize human Zip8 and remarkably demonstrate that upregulation of Zip8 is sufficient to protect lung epithelia against TNF-[alpha]-induced cytotoxicity. We conclude that Zip8 is unique, relative to other Zip proteins, by functioning as an essential zinc importer at the onset of inflammation, thereby facilitating cytoprotection within the lung. inflammation; tissue injury; cell death

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-[1.sup.+/+]) and HSF-1-knockout (HSF-[1.sup.-/-]) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-[alpha] (PFT-[alpha]) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-[1.sup.+/+] cells, PFT-[alpha] attenuated Dox-induced toxicity. However, in HSF-[1.sup.-/-] cells (which express a very low level of HSP27 compared with HSF-[1.sup.+/+] cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-[1.sup.+/+] and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in [G.sub.2]/M phase cell cycle arrest in Doxtreated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death. redox signaling; DNA repair; cell survival; oxidative stress

Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100-F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4-5 days, it was nearly undetectable. Early induction of autophagosomes was associated with their fusion with lysosomes, a phenomenon that later became subverted. Autophagic cell death can be triggered by the products of damaged plasma membrane, sphingolipids, and ceramide. We observed a clustering of membrane rafts shortly after exposure to GC; later, after 24 h, we observed an internalization, accompanied by an increased acid sphingomyelinase activity and accumulation of ceramide. Pharmacological inhibition of autophagy prevented development of premature senescence but did lead to the enhanced rate of apoptosis in human umbilical vein endothelial cells exposed to GC. Pharmacological induction of autophagy resulted in reciprocal changes. These observations appear to represent a mechanistic molecular cascade whereby advanced glycation end products like GC induce sphingomyelinase activity, accumulation of ceramide, clustering, and later internalization of lipid rafts. advanced glycation end product; lysosomes; sphingomyelinase activity; lipid rafts

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyp is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by [beta]-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyp in mitochondrial function of mammalian cells. mitochondria | permeability transition | polyphosphate | [beta]-amyloid peptide | necrosis

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder. HD is caused by polyglutamine expansions in the huntingtin (htt) protein that result in neuronal loss and contribute to HD pathology. The mechanisms of neuronal loss in HD are elusive, and there is no therapy to alleviate HD. To find small molecules that slow neuronal loss in HD, we screened 1,040 biologically active molecules to identify suppressors of cell death in a neuronal cell culture model of HD. We found that inhibitors of mitochondrial function or glycolysis rescued cell death in this cell culture and in in vivo HD models. These inhibitors prevented cell death by activating prosurvival ERK and AKT signaling but without altering cellular ATP levels. ERK and AKT inhibition through the use of specific chemical inhibitors abrogated the rescue, whereas their activation through the use of growth factors rescued cell death, suggesting that this activation could explain the protective effect of metabolic inhibitors. Both ERK and AKT signaling are disrupted in HD, and activating these pathways is protective in several HD models. Our results reveal a mechanism for activating prosurvival signaling that could be exploited for treating HD and possibly other neurodegenerative disorders. caspase | ERK | survival signaling | drugs | neurodegeneration

Whereas NF-[kappa]B has potent antiapoptotic function in most cell types, it was reported that in pancreatic [beta] cells it serves a proapoptotic function and may contribute to the pathogenesis of autoimmune type 1 diabetes. To investigate the role of [beta] cell NF-[kappa]B in autoimmune diabetes, we produced transgenic mice expressing a nondegradable form of IKB[a in pancreatic [beta] cells (RIP-ml[kappa]B[alpha] mice). [beta] cells of these mice were more susceptible to killing by TNF-[alpha] plus IFN-[gamma], but more resistant to IL-1 [beta] plus IFN-[gamma], than normal [beta] cells. Similar results were obtained with [beta] cells lacking I[kappa]B kinase [beta], a protein kinase required for NF-[kappa]B activation. Inhibition of [beta] cell NF-[kappa]B accelerated the development of autoimmune diabetes in nonobese diabetic mice but had no effect on glucose tolerance or serum insulin in C57BL/6 mice, precluding a nonphysiological effect of transgene expression. Development of diabetes after transfer of diabetogenic CD[4.sup.+] T cells was accelerated in RIP-ml[kappa]B[alpha]/nonobese diabetic mice and was abrogated by anti-TNF therapy. These results suggest that under conditions that resemble autoimmune type 1 diabetes, the dominant effect of NF-[kappa]B is prevention of TNF-induced apoptosis. This differs from the proapoptotic function of NF-[kappa]B in IL-1 [beta]-stimulated [beta] cells.

Naturally occurring cell death is a universal feature of developing nervous systems that plays an essential role in determining adult brain function. Yet little is known about the decisions that select a subset of CNS neurons for survival and cause others to die. We report that postnatal day 0 NMDA receptor subunit 1 (NMDAR1) knockout mice display an [approximately equal to]2-fold increase in cell death in the brainstem trigeminal complex (BSTC), including all four nuclei that receive somatosensory inputs from the face (principalis, oralis, interpolaris, and caudalis). Treatment with the NMDA receptor antagonist dizocilpine maleate (MK-801) for 24 h before birth also caused an increase in cell death that reached statistical significance in two of the four nuclei (oralis and interpolaris). The neonatal sensitivity to NMDA receptor hypofunction in the BSTC, and in its main thalamic target, the ventrobasal nucleus (VB), coincides with the peak of naturally occurring cell death and trigeminothalamic synaptogenesis. At embryonic day 17.5, before the onset of these events, NMDAR1 knockout does not affect cell survival in either the BSTC or the VB. Immunostaining for active caspase-3 and the neuronal marker Hu specifically confirms the presence of dying neurons in the BSTC and the VB of NMDAR1 knockout neonates. Finally, genetic deletion of Bax rescues these structures from the requirement for NMDA receptors to limit naturally occurring cell death. Taken together, the results indicate that NMDA receptors play a survival role for somatosensory relay neurons during synaptogenesis by inhibiting Bax-dependent developmental cell death. brainstem | neuroprotection | sensory systems | trophic | ventrobasal

Therapeutic agents acting via cell death and cell protective pathways are becoming increasingly prominent. Prevention of cell death is of importance in stroke, myocardial infarction, and neurodegenerative conditions, while highly selective augmentation of cell death will be sought in anticancer drugs.

The stress-activated protein kinase p38 and nitric oxide (NO) are proposed downstream effectors of excitotoxic cell death. Although the postsynaptic density protein PSD95 can recruit the calcium-dependent neuronal NO synthase (nNOS) to the mouth of the calcium-permeable NMDA receptor, and depletion of PSD95 inhibits excitotoxicity, the possibility that selective uncoupling of nNOS from PSD95 might be neuroprotective is unexplored. The relationship between excitotoxic stress-generated NO and activation of p38, and the significance of the PSD95-nNOS interaction to p38 activation also remain unclear. We find that NOS inhibitors reduce both glutamate-induced p38 activation and the resulting neuronal death, whereas NO donor has effects consistent with NO as an upstream regulator of p38 in glutamate-induced cell death. Experiments using a panel of decoy constructs targeting the PSD95-nNOS interaction suggest that this interaction and subsequent NO production are critical for glutamate-induced p38 activation and the ensuing cell death, and demonstrate that the PSD95-nNOS interface provides a genuine possibility for design of neuroprotective drugs with increased selectivity.

Skeletal muscle in congestive heart failure is responsible for increased fatigability and decreased exercise capacity. A specific myopathy with increased expression of fast-type myosins, myocyte atrophy, secondary to myocyte apoptosis triggered by high levels of circulating tumor necrosis factor-[alpha] (TNF-[alpha]) has been described. In an animal model of heart failure, the monocrotaline-treated rat, we have observed an increase of apoptotic skeletal muscle nuclei. Proapoptotic agents, caspase-3 and -9, were increased, as well as serum levels of TNF-[alpha] and its second messenger sphingosine. Treatment of rats with L-carnitine, known for its protective effect on muscle metabolism injuries, was found to inhibit caspases and to decrease the levels of TNF-[alpha] and sphingosine, as well as the number of apoptotic myonuclei. Staurosporine was used in in vitro experiments to induce apoptosis in skeletal muscle cells in culture. When L-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that L-carnitine can prevent apoptosis of skeletal muscles cells and has a role in the treatment of congestive heart failure-associated myopathy. tumor necrosis factor; sphingosine; caspases; staurosporine

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with [alpha]-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-[x.sub.L] and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c. polyamines; [alpha]-difluoromethylornithine; mitochondria; Bcl-2 protein; caspase-9

The X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. [??]RING point mutants of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that lAP antagonism is required for apoptosis to proceed following UV irradiation.

Results indicate a bifunctional role for the cydAB operon products like preventing the buildup of oxidative free radicals and intracellular detoxification, which facilitate cell suvival and virulence in Brucella abortus.

Cell death inhibition is discussed in this mini-review article, which emphasizes keeping caspases in check relative to apoptosis. Mechanisms of the direct caspase inhibition are the focus, amplified with a description of the structure of a viral inhibitor in complex with a caspase.

A range of molecules control nerve-cell survival in the brain. Many of these molecules might be neuroprotective through activation of the transcription factor cAMP-response-element-binding protein (CREB). Activation of CREB, by phosphorylation of Ser 133, occurs in brain-damage-resistant hippocampal dentate granule cells and is triggered by neuroprotective environmental stimulation. In addition, the Akt neuroprotective signaling pathway activates CREB, and CREB synthesis and phosphorylation promote the survival of many cells, including neurons, in vitro. Thus, CREB might be responsible for programmed nerve-cell survival. Studies investigating its role in the brain are now required to confirm these in vitro results, and the downstream survival genes, whose expression is activated by CREB in neurons, need to be identified.

Tumor necrosis factor a (TNF-[Alpha]) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription factor nuclear factor kappa B (NF-ΚB), which suppresses apoptosis by an unknown mechanism. The activation of NF-ΚB was found to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 were identified as gene targets of NF-ΚB transcriptional activity. In cells in which NF-ΚB was inactive, all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to suppress etoposide-induced apoptosis. Thus, NF-ΚB activates a group of gene products that function cooperatively at the earliest checkpoint to suppress TNF-[Alpha]-mediated apoptosis and that function more distally to suppress genotoxic agent-mediated apoptosis., TNF engagement of TNFR1 leads to the recruitment of TRADD (TNFR1-associated death domain protein) and RIP (receptor-interacting protein) to the receptor complex (1). TRADD interacts with FADD (Fas-associated death domain [...]

The effects of insulin-like growth factor I (IGF-I) on the survival and regeneration of lymphocytes in the thymus and spleen were investigated in rats treated with dexamethasone and control subjects. Results show that IGF-I reduced lymphocyte apoptosis in the spleen but not in the thymus. In control animals, IGF-I increased the total number of lymphocytes. These results indicate that IGF-I modulates immune homeostasis by influencing the survival and generation of lymphocytes.

Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor. Herein prosaposin was found to increase sulfatide concentrations in primary and transformed Schwann cells (iSC) and oligodendrocytes (differentiated CG4 cells). Of the four mature saposins, only saposin C was found to increase sulfatide concentrations in these cell types. A similar result was obtained by using peptides (prosaptides) encompassing the neurotrophic sequence located in the saposin C domain. Dose-response curves demonstrated maximal enhancement by saposin C and prosaptides at low nanomolar concentrations (5-10 nM). The increase in sulfatide concentration by a 14-mer prosaptide, TX14(A), in CG4 oligodendrocytes was about 3-fold greater than in primary Schwann cells. A mutant prosaptide with a single amino acid replacement of Asn [approaches] Asp was inactive. Prosaptides did not induce cell proliferation of primary Schwann cells, iSC cells, or CG4 oligodendrocytes but nanomolar concentrations of prosaptides prevented cell death of iSC cells and CG4 oligodendrocytes. Immunoblot analysis demonstrated that phosphorylation of both mitogen-activated protein kinase p-42 and p-44 isoforms were enhanced 3- to 5-fold after 5 min of treatment with prosaptides at concentrations of 1-5 nM. These findings suggest that prosaposin and prosaptides bind to a receptor that initiates signal transduction to promote myelin lipid synthesis and prolong cell survival in both Schwann cells and oligodendrocytes. Prosaposin may function as a myelinotrophic factor in vivo during development and repair of myelinated nerves explaining the deficiency of myelin observed in prosaposin-deficient mice and humans.

Glial cell line-derived neutrotrophic factor (GDNF) protects injured adult mammalian motoneurons from cell death in vivo thereby indicating that GDNF is suitable for therapeutic inhibition of cell loss after spinal cord injury and in the diagnosis of motoneuron diseases. A mouse model of injury initiated adult motoneuron degeneration is used to analyze the trophic influence of GDNF. All the motoneurons are eliminated after 6 weeks following surgery due to the injury at the exit point of the nerve from spinal cord. GDNF induces an increase in the surviving ability of the motoneurons.

N-acetyl-L-cysteine (NAC) is a pluripotent protector against cell death due to cytotoxic stimuli and increases the survival of cells with low concentrations of trophic factors. NAC inhibits tumor necrosis factor alpha-induced deaths of oligodendrocytes and L929 fibroblasts and blocks glutamate initiated oligodendrocyte death. Various physiological mechanisms explain the prevention of cell death and presence of activators of tyrosine kinase activity is not necessary for prevention.

An analysis of the reduction of apoptosis in cerebellar granule neurons by low potassium reveals that differentiated granule neurons degenerate and die, when they are shifted from a culture medium with high K+ to one with a low, but more physiological K+ concentration. Apoptosis or programmed cell death is responsible for death by low potassium.

The antioxidant potential of Bcl-2 prevents morphologically patterned cell death or apoptosis. Intracellular production of oxygen free radicals occurs during apoptosis. The characteristic features of apoptosis and the functional role of Bcl-2 during this process provide evidence for the common molecular mechanism involved in cell death. Bcl-2 also safeguards cells from oxidative fatality of stimulation by hydrogen peroxide and menadione. An overly expressed Bcl-2 leads to inactivation of lipid peroxidation. Bcl-2 activity also prevents cell death stimulated by gamma irradiation.

Epstein-Barr virus-coded early lytic cycle protein BHRF1, which shares sequence homology with the cellular survival protein Bcl-2, affects cell viability of infected B cells through the up-regulation of Bcl-2. The probable effects of BHRF1 on cell viability are determined by observing the gene expression encoding protein in BL-cell lines, a prototype of human B-lymphoid. BL-cells are extremely responsive to apoptosis triggered by lack of serum. An examination of 3 BL-cell lines selected for BHRF1 gene transfection elucidate the functional homology between BHRF1 and Bcl-2 in affecting cell viability.

A proportion of developing oligodendrocytes undergo natural cell death by apoptosis, and mature oligodendrocytes die, either by apoptosis or necrosis, in response to injurious signals such as cytotoxic cytokines and complement. Ciliary neurotrophic factor (CNTF), a trophic factor found in astrocytes in the central nervous system (CNS), promoted the survival and maturation of cultured olijodendrocytes. This trophic factor also protected oligodendrocytes from death induced by tumor necrosis factors (apoptosis) but not against complement (necrosis). These results suggest that CNTF functions in the survival of oligodendrocytes during development and may lead to therapeutic approaches for degenerative diseases of the CNS that involve oligodendrocyte destruction., Natural cell death is important in the cell development and cell turnover of higher vertebrates[1]. Natural cell death occurs through an active process that requires RNA and protein synthesis[2]. Cells [...]

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay showed significant increase in cell viability when PC-12 cells were treated with apple extracts. The results suggest that naturally occurring antioxidants, such as phenolic phytochemicals in fresh apples, may reduce the risk of neurodegenerative disorders.

This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the n-3 family, play pivotal roles as 'fuel partitioners' in that they direct fatty acids away from triglyceride storage and toward oxidation and they enhance glucose flux to glycogen. In doing this, PUFA may reduce the risk of enhanced cellular apoptosis associated with excessive cellular lipid accumulation. PUFA exert their beneficial effects by upregulating the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously downregulating genes encoding proteins of lipid synthesis. PUFA govern oxidative gene expression by activating the transcription factor peroxisome proliferator-activated receptor-[Alpha]. PUFA suppress lipogenic gene expression by reducing the nuclear abundance and DNA binding affinity of transcription factors responsible for imparting insulin and carbohydrate control to lipogenic and glycolytic genes. In particular, PUFA suppress the nuclear abundance and expression of sterol regulatory element binding protein-1 and reduce the DNA binding activities of nuclear factor Y, stimulatory protein 1, and possibly hepatic nuclear factor-4. Collectively, the studies discussed suggest that the fuel 'repartitioning' and gene expression actions of PUFA should be considered among the criteria used in defining the dietary needs of n-6 and n-3 fatty acids and in establishing the dietary ratio of n-6 to n-3 fatty acids needed for optimum health benefit. sterol regulatory element binding protein; fatty livers; nuclear factor Y; transcription

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in BcI-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

Mitochondrial membrane potential ([Delta][[Psi].sub.m]) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive [K.sup.+] (mito[K.sub.ATP]) channel activation prevents the collapse of [Delta][[Psi].sub.m] in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed [Delta][[Psi].sub.m], as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mito[K.sub.ATP] channel with diazoxide (100 [micro]mol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized [Delta][[Psi].sub.m]. This protection was blocked by 5-hydroxydecanoate (500 [micro]mol/l), a mito[K.sub.ATP] channel-selective inhibitor, but not by HMR-1098 (30 [micro]mol/l), a putative sarcolemmal [K.sub.ATP] channel-selective inhibitor. Dissipation of [Delta][[Psi].sub.m] also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts [Delta][[Psi].sub.m] and induces apoptosis, which are prevented by the activation of the mito[K.sub.ATP] channel. This further emphasizes the therapeutic significance of mito[K.sub.ATP] channel agonists in the prevention of ischemia-reperfusion cell injury. apoptosis; myocytes; ATP; permeability transition pore; cytochrome c

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/ K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) [Alpha] or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Bcl-2 decreases voltage-gated [K.sup.+] channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C157-C165, 2001.--Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to [K.sup.+] efflux; blockade of plasmalemmal [K.sup.+] channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated [K.sup.+] (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents ([I.sub.Kv]). In contrast, the apoptosis inducer staurosporine (ST) enhanced [I.sub.Kv]. In bcl-2-infected cells, however, the ST-induced increase in [I.sub.Kv] was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in [I.sub.Kv] and apoptesis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of [I.sub.Kv] and downregulated the mRNA expression of the pore-forming Ky channel [Alpha]-subunits (Kv1.1, Kv1.5, and Ky2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells. apoptotic volume decrease; pulmonary artery smooth muscle cells; current density of voltage-gated potassium channels