Fas is a cell surface receptor that controls a poorly understood signal transduction pathway that leads to cell death by means of apoptosis. A protein tyrosine phosphatase, FAP-1, capable of interacting with the cytosolic domain of Fas, was identified. The carboxyl terminal 15 amino acids of Fas are necessary and sufficient for interaction with FAP-1. FAP-1 expression is highest in tissues and cell lines that are relatively resistant to Fas-mediated cytotoxicity. Gene transfer-mediated elevations in FAP-1 partially abolished Fas-induced apoptosis in a T cell line. These findings are consistent with an inhibitory effect of FAP-1 on Fas signal transduction.

Friedreich ataxia (FA) is associated with the expansion of a GAA trinucleotide repeat in the first intron of the X25 gene. We found both alleles expanded in 67 FA patients from 48 Italian families. Five patients from three families were compound heterozygotes with expansion on one allele and an isoleucine->>phenylalanine change at position 154 on the other one. We found neither expansions nor point mutations in three patients. The length of FA alleles ranged from 201 to 1,186 repeat units, with no overlap with the normal range, and showed a negatively skewed distribution with a peak between 800 and 1,000 repeats. The FA repeat showed meiotic instability with a median variation of 150 repeats. The lengths of both larger and smaller alleles in each patient inversely correlated with age at onset of the disorder. Smaller alleles showed the best correlation, accounting for approximately 50% of the variation of age at onset. Mean allele length was significantly higher in patients with diabetes and in those with cardiomyopathy.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B(-/-) mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1(-/-) mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.

The anterior cingulate cortex (ACC) is part of a network implicated in the development of self-regulation and whose connectivity changes dramatically in development. In previous studies we showed that 3 h of mental training, based on traditional Chinese medicine (integrative body-mind training, IBMT), increases ACC activity and improves self-regulation. However, it is not known whether changes in white matter connectivity can result from small amounts of mental training. We here report that 11 h of IBMT increases fractional anisotropy (FA), an index indicating the integrity and efficiency of white matter in the corona radiata, an important white-matter tract connecting the ACC to other structures. Thus IBMT could provide a means for improving self-regulation and perhaps reducing or preventing various mental disorders.

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

The innate immune system uses Toll family receptors to signal for the presence of microbes and initiate host defense. Bacterial lipoproteins (BLPs), which are expressed by all bacteria, are potent activators of Toll-like receptor-2 (TLR2). Here we show that the adaptor molecule, myeloid differentiation factor 88 (MyD88), mediates both apoptosis and nuclear factor-kappaB (NF-kappaB) activation by BLP-stimulated TLR2. Inhibition of the NF-kappaB pathway downstream of MyD88 potentiates apoptosis, indicating that these two pathways bifurcate at the level of MyD88. TLR2 signals for apoptosis through MyD88 via a pathway involving Fas-associated death domain protein (FADD) and caspase 8. Moreover, MyD88 binds FADD and is sufficient to induce apoptosis. These data indicate that TLR2 is a novel 'death receptor' that engages the apoptotic machinery without a conventional cytoplasmic death domain. Through TLR2, BLP induces the synthesis of the precursor of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). Interestingly, BLP also activates caspase 1 through TLR2, resulting in proteolysis and secretion of mature IL-1beta. These results indicate that caspase activation is an innate immune response to microbial pathogens, culminating in apoptosis and cytokine production.

In multicellular organisms, cell death is a critical and active process that maintains tissue homeostasis and eliminates potentially harmful cells. There are three major types of morphologically distinct cell death: apoptosis (type I cell death), autophagic cell death (type II), and necrosis (type III). All three can be executed through distinct, and sometimes overlapping, signaling pathways that are engaged in response to specific stimuli. Apoptosis is triggered when cell-surface death receptors such as Fas are bound by their ligands (the extrinsic pathway) or when Bcl2-family proapoptotic proteins cause the permeabilization of the mitochondrial outer membrane (the intrinsic pathway). Both pathways converge on the activation of the caspase protease family, which is ultimately responsible for the dismantling of the cell. Autophagy defines a catabolic process in which parts of the cytosol and specific organelles are engulfed by a double-membrane structure, known as the autophagosome, and eventually degraded. Autophagy is mostly a survival mechanism; nevertheless, there are a few examples of autophagic cell death in which components of the autophagic signaling pathway actively promote cell death. Necrotic cell death is characterized by the rapid loss of plasma membrane integrity. This form of cell death can result from active signaling pathways, the best characterized of which is dependent on the activity of the protein kinase RIP3.

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.

Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.

Spontaneous apoptosis was observed in a proportion of peripheral blood mononuclear cells obtained from patients with head and neck cancer (HNC) but not from normal healthy donors (T. Saito et al., Clin. Cancer Res., 5: 1263-1273, 1999). To further investigate this phenomenon, peripheral blood mononuclear cells were obtained from patients with HNC or normal controls (NCs) and evaluated for expression of apoptosis markers (annexin V binding and caspase-3 activation), T-cell receptor-associated zeta chain, and the death receptor Fas (APO-1, CD95) in CD3(+) T cells by multicolor flow cytometry. Soluble Fas ligand (sFasL) in the sera of these individuals was quantitated by ELISA. In patients with HNC, 74 +/- 15% (mean +/- SD) of CD3(+) T cells were Fas(+) compared with 52 +/- 13% in NCs (P < 0.0001). Furthermore, 29 +/- 16% of the Fas(+) CD3(+) T cells bound annexin V in patients and only 14% +/- 7% of the Fas(+) CD3(+) T cells bound annexin V in NCs (P < 0.0001). In patients, Fas(+) CD3(+) cells preferentially underwent apoptosis and showed a loss of zeta chain expression. Significantly greater proportions of CD8(+) T cells than CD4(+) T cells were apoptotic (P < 0.0002), which indicates that CD8(+) T cells were especially sensitive to apoptosis. Serum levels of sFasL were lower in HNC patients with active disease than in NCs or in patients with no evident disease (P < 0.0183). This suggested utilization of sFasL produced in vivo and activation of the Fas/Fas ligand (FasL) pathway in Fas(+) T cells. Proportions of apoptotic T cells were higher in HNC patients than in NCs (P < 0.0001), and a subset of HNC patients with active disease had the highest proportions of circulating Fas(+) annexin V(+) T lymphocytes. The data indicate that the Fas/FasL pathway is involved in spontaneous apoptosis of circulating Fas(+) T lymphocytes in cancer patients. Fas/FasL interactions might lead to excessive turnover of T cells in the circulation and, consequently, to reduced immune competence in patients with HNC.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Young's moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

The tumor suppressor protein p53 regulates transcriptional programs that control the response to cellular stress. We show that distinct mechanisms exist to activate p53 target genes as revealed by marked differences in affinities and damage-specific recruitment of transcription initiation components. p53 functions in a temporal manner to regulate promoter activity both before and after stress. Before DNA damage, basal levels of p53 are required to assemble a poised RNA polymerase II initiation complex on the p21 promoter. RNA pol II is converted into an elongating form shortly after stress but before p53 stabilization. Proapoptotic promoters, such as Fas/APO1, have low levels of bound RNA pol II but undergo damage-induced activation through efficient reinitiation. Surprisingly, in a p53-dependent process key basal factors TAFII250 and TFIIB assemble into the transcription machinery in a stress- and promoter-specific manner, behaving as differential cofactors for p53 action after distinct types of DNA damage.

MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL.

Cholangiocarcinomas are devastating cancers that are increasing in both their worldwide incidence and mortality rates. The challenges posed by these often lethal biliary tract cancers are daunting, with conventional treatment options being limited and the only hope for long-term survival being that of complete surgical resection of the tumor. Unfortunately, the vast majority of patients with cholangiocarcinoma typically seek treatment with advanced disease, and often these patients are deemed poor candidates for curative surgery. Moreover, conventional chemotherapy and radiation therapy have not been shown to be effective in prolonging long-term survival, and although photodynamic therapy combined with stenting has been reported to be effective as a palliative treatment, it is not curative. Thus, there is a real need to develop novel chemopreventive and adjuvant therapeutic strategies for cholangiocarcinoma based on exploiting select molecular targets that would impact in a significant way on clinical outcome. This review focuses on potential preventive targets in cholangiocarcinogenesis, such as inducible nitric oxide synthase, cyclooxygenase-2, and altered bile acid signaling pathways. In addition, molecular alterations related to dysregulation of cholangiocarcinoma cell growth and survival, aberrant gene expression, invasion and metastasis, and tumor microenvironment are described in the context of various clinical and pathological presentations. Moreover, an emphasis is placed on the importance of critical signaling pathways and postulated interactions, including those of ErbB-2, hepatocyte growth factor/Met, interleukin-6/glycoprotein130, cyclooxygenase-2, vascular endothelial growth factor, transforming growth factor-beta, MUC1 and MUC4, beta-catenin, telomerase, and Fas pathways as potential molecular therapeutic targets in cholangiocarcinoma.

Diffusion tensor magnetic resonance imaging (DTI) was used to study regional changes in the brain's development from childhood (8-12 years, mean 11.1 +/- 1.3, N = 32) to young adulthood (21-27 years, mean 24.4 +/- 1.8, N = 28). Mean diffusivity (Trace/3 apparent diffusion coefficient, ADC) and fractional anisotropy (FA) were measured in 30 regions of interest (ROIs) in 13 distinct brain structures. Correlational analysis was performed to detect changes within 8-12 years and within 21-27 years, and group analysis to compare childhood diffusion properties with young adult values. Increases of fractional anisotropy were seen in the genu of the corpus callosum, splenium of the corpus callosum, corona radiata, putamen, and head of the caudate nucleus within 8-12 years, and also between childhood and young adulthood. Reductions in Trace/3 ADC were observed in 9 of 13 structures within 8-12 years and into young adulthood as well. DTI demonstrates more widespread changes in the brain's microstructure with maturation than previous reports using conventional T1-weighted MRI scans. These findings suggest a continuation of the brain's microstructural development through adolescence.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.

During development, neuronal survival is regulated by the limited availability of neurotrophins, which are proteins of the nerve growth factor (NGF) family. Activation of specific trk tyrosine kinase receptors by the neurotrophins blocks programmed cell death. The trkA-specific ligand NGF has also been shown to activate the non-tyrosine kinase receptor p75, a member of the tumour necrosis factor (TNF) receptor and Fas (APO-1/CD95) family. Here we report that, early in development, endogenous NGF causes the death of retinal neurons that express p75 but not trkA. These results indicate that, as with cells of the immune system, the death of neurons in the central nervous system can also be induced by ligands, and that the effect of NGF on cell fate depends on the type of receptor expressed by developing neurons.

Once osteoblasts have completed their bone-forming function, they are either entrapped in bone matrix and become osteocytes or remain on the surface as lining cells. Nonetheless, 50-70% of the osteoblasts initially present at the remodeling site cannot be accounted for after enumeration of lining cells and osteocytes. We hypothesized that the missing osteoblasts die by apoptosis and that growth factors and cytokines produced in the bone microenvironment influence this process. We report that murine osteoblastic MC3T3-E1 cells underwent apoptosis following removal of serum, or addition of tumor necrosis factor (TNF), as indicated by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and DNA fragmentation studies. Transforming growth factor-beta and interleukin-6 (IL-6)-type cytokines had antiapoptotic effects because they were able to counteract the effect of serum starvation or TNF. In addition, anti-Fas antibody stimulated apoptosis of human osteoblastic MG-63 cells and IL-6-type cytokines prevented these changes. The induction of apoptosis in MG-63 cells was associated with an increase in the ratio of the proapoptotic protein bax to the antiapoptotic protein bcl-2, and oncostatin M prevented this change. Examination of undecalcified sections of murine cancellous bone revealed the presence of apoptotic cells, identified as osteoblasts by their proximity to osteoid seams and their juxtaposition to cuboidal osteoblasts. Assuming an osteoblast life span of 300 h and a prevalence of apoptosis of 0.6%, we calculated that the fraction that undergo this process in vivo can indeed account for the missing osteoblasts. These findings establish that osteoblasts undergo apoptosis and strongly suggest that the process can be modulated by growth factors and cytokines produced in the bone microenvironment.

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GST)-TRAIL extracellular domain fusion proteins were produced to analyze TRAIL-induced apoptosis. Only GST-TRAIL constructs containing regions homologous to the Fas self-association and ligand binding domains could induce apoptosis. GST-TRAIL induced significant (>90%) apoptosis in just one of eight normal and one of eight malignant breast cell lines. All other lines were relatively resistant to TRAIL-induced apoptosis. Activating TRAIL receptors DR4 and DR5 were expressed in all normal and malignant breast cell lines. The inhibitory receptor TRID was highly expressed in one of four normal and two of seven malignant breast cell lines. DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of cell lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-induced apoptosis in most breast cell lines. By fractional inhibition analysis, the toxicity of the combination of TRAIL and doxorubicin or 5-fluorouracil was synergistic compared with either agent alone. In contrast, melphalan and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and methotrexate did not augment it in any cell line. Augmentation of TRAIL-induced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation. This was evidenced by the fact that chemotherapy agents that synergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocked by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk). The combination of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In contrast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e.g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk. These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. In summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no difference in sensitivity is found between normal and malignant cell lines. Treatment with chemotherapy provides an approach to sensitize breast cancer cells to TRAIL-induced apoptosis.

We analyzed the potential causes of the increased susceptibility to apoptosis of peripheral lymphocytes from a large cohort of HIV-infected persons that we followed during a 3-yr period. By using quantitative cytofluorometric methods, we demonstrate that all lymphocyte populations were contributing proportionally to the apoptotic population in both groups of donors, but the percentage of T and B cells involved in this cell death process was significantly increased in HIV-infected persons. To study the relationship between the increased apoptosis in HIV infection and the activation state of the immune system, we analyzed cell surface expression of activation markers on apoptotic and nonapoptotic cells. We found that in the chronic phase of HIV infection, 50 to 60% of the apoptotic cells exhibited an activated phenotype (they were HLA-DR+, CD38+, CD45RO+, and Fas+), and interestingly, the CD45RO+ subset appeared to be more prone to apoptosis in HIV-positive persons. This study also indicates that the activated phenotype found on apoptotic cells was not a distinctive feature of patients' lymphocytes since it was in similar proportion in apoptotic cells from control lymphocytes. However, a significant correlation was found between the intensity of anti-CD3-induced apoptosis in both CD4 and CD8 T cells from HIV-infected persons and their in vivo expression of CD45RO and HLA-DR molecules. Finally, a significant correlation was found between the intensity of spontaneous or anti-CD3-induced apoptosis in total lymphocytes and disease progression; this was confirmed when the CD4 and CD8 T cell subsets were analyzed separately. Altogether these observations indicate that the increased susceptibility to apoptosis of peripheral T cells from HIV-infected persons correlates with disease progression and support the hypothesis that the chronic activation of the immune system occurring throughout HIV infection is the primary mechanism responsible for this cell deletion process.

Fatty acids (FAs) and other lipid molecules are important for many cellular functions, including vesicle exocytosis. For the pancreatic beta-cell, while the presence of some FAs is essential for glucose-stimulated insulin secretion, FAs have enormous capacity to amplify glucose-stimulated insulin secretion, which is particularly operative in situations of beta-cell compensation for insulin resistance. In this review, we propose that FAs do this via three interdependent processes, which we have assigned to a "trident model" of beta-cell lipid signaling. The first two arms of the model implicate intracellular metabolism of FAs, whereas the third is related to membrane free fatty acid receptor (FFAR) activation. The first arm involves the AMP-activated protein kinase/malonyl-CoA/long-chain acyl-CoA (LC-CoA) signaling network in which glucose, together with other anaplerotic fuels, increases cytosolic malonyl-CoA, which inhibits FA partitioning into oxidation, thus increasing the availability of LC-CoA for signaling purposes. The second involves glucose-responsive triglyceride (TG)/free fatty acid (FFA) cycling. In this pathway, glucose promotes LC-CoA esterification to complex lipids such as TG and diacylglycerol, concomitant with glucose stimulation of lipolysis of the esterification products, with renewal of the intracellular FFA pool for reactivation to LC-CoA. The third arm involves FFA stimulation of the G-protein-coupled receptor GPR40/FFAR1, which results in enhancement of glucose-stimulated accumulation of cytosolic Ca2+ and consequently insulin secretion. It is possible that FFA released by the lipolysis arm of TG/FFA cycling is partly "secreted" and, via an autocrine/paracrine mechanism, is additive to exogenous FFAs in activating the FFAR1 pathway. Glucose-stimulated release of arachidonic acid from phospholipids by calcium-independent phospholipase A2 and/or from TG/FFA cycling may also be involved. Improved knowledge of lipid signaling in the beta-cell will allow a better understanding of the mechanisms of beta-cell compensation and failure in diabetes.

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.

The Fas/APO-1 receptor is one of the major regulators of apoptosis. We report here that Fas/APO-1-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-1 beta-converting enzyme (ICE), which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/APO-1 rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/APO-1-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor CrmA or an antisense ICE construct, substantially suppressed Fas/APO-1-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/APO-1-mediated cell death.

The integrity of white matter, as measured in vivo with diffusion tensor imaging (DTI), is disrupted in normal aging. A current consensus is that in adults advancing age affects anterior brain regions disproportionately more than posterior regions; however, the mainstay of studies supporting this anterior-posterior gradient is based primarily on measures of the corpus callosum. Using our quantitative fiber tracking approach, we assessed fiber tract integrity of samples of major white matter cortical, subcortical, interhemispheric, and cerebellar systems (11 bilateral and 2 callosal) on DTI data collected at 1.5T magnet strength. Participants were 55 men (age 20-78 years) and 65 women (age 28-81 years), deemed healthy and cognitively intact following interview and behavioral testing. Fiber integrity was measured as orientational diffusion coherence (fractional anisotropy, FA) and magnitude of diffusion, which was quantified separately for longitudinal diffusivity (lambdaL), an index of axonal length or number, and transverse diffusivity (lambdaT), an index of myelin integrity. Aging effects were more evident in diffusivity than FA measures. Men and women, examined separately, showed similar age-related increases in longitudinal and transverse diffusivity in fibers of the internal and external capsules bilaterally and the fornix. FA was lower and diffusivity higher in anterior than posterior fibers of regional paired comparisons (genu versus splenium and frontal versus occipital forceps). Diffusivity with older age was generally greater or FA lower in the superior than inferior fiber systems (longitudinal fasciculi, cingulate bundles), with little to no evidence for age-related degradation in pontine or cerebellar systems. The most striking sex difference emerged for the corpus callosum, for which men showed significant decline in FA and increase in longitudinal and transverse diffusivity in the genu but not splenium. By contrast, in women the age effect was present in both callosal regions, albeit modestly more so in the genu than splenium. Functional meaningfulness of these age-related differences was supported by significant correlations between DTI signs of white matter degradation and poorer performance on cognitive or motor tests. This survey of multiple fiber systems throughout the brain revealed a differential pattern of age's effect on regional FA and diffusivity and suggests mechanisms of functional degradation, attributed at least in part to compromised fiber microstructure affecting myelin and axonal morphology.