In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

The purpose of this study is to elucidate changes in mean diffusivity (ADC) and fractional anisotropy (FA) using MR diffusion tensor imaging (DTI) in the central nervous system during normal aging. We studied 50 normal volunteers (30 men, 20 women; mean age 44.8 +/- 14.0; age range, 21-69 years) without disorders affecting the central nervous system. The frontal, parietal white matter, lentiform nucleus, posterior limb of internal capsule, thalamus, genu and splenium of the corpus callosum were selected for investigation. There was no significant difference in ADC or FA between male and female or between the right and left hemisphere. A significant ADC increase with advancing age was observed in frontal white matter (P = 0.010) and lentiform nucleus (P = 0.022). A significant FA decline was found only in the genu of the corpus callosum (P < 0.001) with advancing age. Quantitative diffusion tensor analysis correlate with normal aging and may help in assessing normal age-related changes and serve as a standard for comparison with neurodegenerative disorders.

The local pattern of proinflammatory cytokine release was studied in Alzheimer disease (AD) and vascular dementia (VAD), by measuring intrathecal levels of IL-1 beta, IL-6, TNF-alpha, and its naturally occurring antagonists, soluble TNF receptors I and II. The cytokine levels were related to neuronal damage, as measured by the intrathecal tau concentration, to cerebral apoptosis assessed by levels of Fas/APO-1 and bcl-2, and to clinical variables. In vitro analysis was performed to study the effect of TNF-alpha on the production of bcl-2, an antiapoptotic factor, by human neuronal cells. Patients with both AD and VAD displayed significantly higher intrathecal levels of TNF-alpha compared to controls. In addition, patients with AD showed significantly negative correlations between the intrathecal levels of TNF-alpha and the levels of Fas/APO-1 as well as of tau protein. The level of bcl-2 in supernatants of TNF-alpha-exposed cultures of human neuronal cells was up to three times higher than in control supernatants. Our study demonstrates intrathecal production of TNF-alpha in patients with dementias, suggesting that this cytokine may have a neuroprotective role in these neurodegenerative conditions as evidenced by negative correlations between this cytokine and (i) levels of intrathecal Fas/APO-1 and (ii) levels of tau protein, both parameters closely related to brain damage. Our in vitro data suggest that TNF-alpha exerts its neuroprotective effect by stimulating neuronal cells to express bcl-2, a molecule which downregulates apoptosis.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.

Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. A total of 13 FA proteins are involved in regulating genome surveillance and chromosomal stability. The FA core complex, consisting of 8 FA proteins (A/B/C/E/F/G/L/M), is essential for the monoubiquitination of FANCD2 and FANCI. FANCM is a human ortholog of the archaeal DNA repair protein Hef, and it contains a DEAH helicase and a nuclease domain. Here, we examined the effect of FANCM expression on the integrity and localization of the FA core complex. FANCM was exclusively localized to chromatin fractions and underwent cell cycle-dependent phosphorylation and dephosphorylation. FANCM-depleted HeLa cells had an intact FA core complex but were defective in chromatin localization of the complex. Moreover, depletion of the FANCM binding partner, FAAP24, disrupted the chromatin association of FANCM and destabilized FANCM, leading to defective recruitment of the FA core complex to chromatin. Our results suggest that FANCM is an anchor required for recruitment of the FA core complex to chromatin, and that the FANCM/FAAP24 interaction is essential for this chromatin-loading activity. Dysregulated loading of the FA core complex accounts, at least in part, for the characteristic cellular and developmental abnormalities in FA.

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.

Interleukin 6 (IL6) trans-signaling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL6 trans-signaling is reliant upon the release of soluble IL6 receptor (sIL6R), which binds IL6 to create an agonistic IL6/sIL6R complex capable of activating cell types that would not normally respond to IL6 itself. Here we show that intrinsic and extrinsic apoptotic stimulation by DNA damage, cytokine deprivation, and Fas stimulation promotes shedding of sIL6R. Apoptosis-induced shedding of the IL6R was caspase dependent but PKC independent, with inhibition of ADAM17 preventing IL6R shedding. Such insight is relevant to the control of acute inflammation, where transition from the initial neutrophil infiltration to a more sustained population of mononuclear cells is essential for the resolution of the inflammatory process. This transitional event is governed by IL6 trans-signaling. This study demonstrates that IL6R is shed from apoptotic human neutrophils. In vivo studies in a murine inflammation model showed that neutrophil depletion resulted in reduced local sIL6R levels and a concomitant decrease in mononuclear cells, suggesting that apoptosis-induced IL6R shedding from neutrophils promotes IL6 trans-signaling and regulates the attraction of monocytic cells involved in the clearance of apoptotic neutrophils.

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.

OBJECTIVE

Major depressive disorder (MDD) occurs frequently in adolescents, but the neurobiology of depression in youth is poorly understood. Structural neuroimaging studies in both adult and pediatric populations have implicated frontolimbic neural networks in the pathophysiology of MDD. Diffusion tensor imaging (DTI), which measures white matter (WM) microstructure, is a promising tool for examining neural connections and how they may be abnormal in MDD.

METHODS

We used two separate approaches to analyze DTI data in adolescents with MDD (n = 14) compared with healthy volunteers (n = 14).

RESULTS

The first, hypothesis-driven approach was to use probabilistic tractography to delineate tracts arising from the subgenual anterior cingulate cortex (ACC). Adolescents with MDD demonstrated lower fractional anisotropy (FA) in the WM tract connecting subgenual ACC to amygdala in the right hemisphere. The second, exploratory approach was to conduct a voxelwise comparison of FA. This analysis revealed 10 clusters where adolescents with MDD had significantly lower (uncorrected) FA than the healthy group within WM tracts including right and left uncinate and supragenual cingulum.

CONCLUSIONS

These preliminary data support the hypothesis that altered WM microstructure in frontolimbic neural pathways may contribute to the pathophysiology of MDD in adolescents.

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.

A number of biochemical defects have been identified in glucose metabolism within skeletal muscle in obesity, and positive effects of weight loss on insulin resistance are also well established. Less is known about the capacity of skeletal muscle for the metabolism of fatty acids in obesity-related insulin resistance and of the effects of weight loss, though it is evident that muscle contains increased triglyceride. The current study was therefore undertaken to profile markers of human skeletal muscle for fatty acid metabolism in relation to obesity, in relation to the phenotype of insulin-resistant glucose metabolism, and to examine the effects of weight loss. Fifty-five men and women, lean and obese, with normal glucose tolerance underwent percutaneous biopsy of vastus lateralis skeletal muscle for determination of HADH, CPT, heparin-releasable (Hr) and tissue-extractable (Ext) LPL, CS, COX, PFK, and GAPDH enzyme activities, and content of cytosolic and plasma membrane FABP. Insulin sensitivity was measured using the euglycemic clamp method. DEXA was used to measure FM and FFM. In skeletal muscle of obese individuals, CPT, CS, and COX activities were lower while, conversely, they had a higher or similar content of FABP(C) and FABP(PM) than in lean individuals. Hr and Ext LPL activities were similar in both groups. In multivariate and simple regression analyses, there were significant correlations between insulin resistance and several markers of FA metabolism, notably, CPT and FABP(PM). These data suggest that in obesity-related insulin resistance, the metabolic capacity of skeletal muscle appears to be organized toward fat esterification rather than oxidation and that dietary-induced weight loss does not correct this disposition.

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

BACKGROUND

Mesial temporal lobe epilepsy (mTLE) with hippocampus sclerosis (HS) is an important cause for focal epilepsy. In this study, we explored the integrity of connecting networks using diffusion tensor imaging (DTI) and two whole-brain voxel-based methods: statistical parametric mapping (SPM) and tract-based spatial statistics (TBSS).

METHODS

Thirty-three consecutive patients with mTLE and HS undergoing presurgical evaluation were scanned at 3 T, a DTI data set was acquired and parametric maps of fractional anisotropy (FA) and mean diffusivity (MD) were calculated. Twenty-one patients had left hippocampal sclerosis (LHS) and 12 patients had right HS (RHS). These groups were compared to 37 normal control subjects using both SPM5 and TBSS.

RESULTS

The ipsilateral temporal lobe showed widespread FA reduction in both groups. The limbic system was clearly abnormal in the LHS group, also involving the arcuate fasciculus. In RHS, changes were more restricted but also showed involvement of the contralateral temporal and inferior frontal lobe. Increased MD was found in the ipsilateral hippocampus by SPM that was only marginally detected by TBSS. In white matter regions, however, TBSS was more sensitive to changes than SPM.

CONCLUSIONS

DTI detects extensive changes in mTLE with HS. The affected networks were principally in the ipsilateral temporal lobe and the limbic system but also the arcuate fasciculus. SPM and TBSS gave complementary information with higher sensitivity to FA changes using TBSS.

The role of lymphocyte apoptosis in septic shock remains a controversial issue. Using Annexin V and flow cytometry analysis on freshly isolated cells, we evaluated circulating lymphocyte apoptosis in 23 septic shock, 25 sepsis without shock, 7 nonseptic critically ill, and 25 control patients. In patients with sepsis, we compared day 1 lymphocyte apoptosis (i.e., within 3 days of the onset of infection) with that observed 5-7 days after (day 6) according to shock state, mortality, and seventy factors. At day 1, patients in septic shock exhibited higher lymphocyte apoptosis than that present in controls (16.5% +/- 3.5% vs. 3% +/- 0.5%, respectively, P = 0.0001). At day 6, patients with sepsis without shock restored undamaged CD4+ T and CD8+ T lymphocyte counts, whereas patients in septic shock increased only CD4+ T cells. Similarly, survivors restored undamaged lymphocyte count at day 6 (+70%, P < 0.001), whereas nonsurvivors did not. Day 6 undamaged lymphocyte count negatively correlated with day 1 SAPS II, day 6 LOD score, mechanical ventilation, and ICU stay duration. We observed no apoptotic effect of septic shock plasma or septic shock circulating mononuclear cells on target lymphoid cell lines. We found no alteration in any death receptors Fas, TRAIL-R1, TRAIL-R2, or in their ligands on circulating blood cells. Catecholamines and interleukin 10 levels significantly increased in patients with septic shock, but did not correlate with apoptosis levels. We conclude that lymphocyte apoptosis is rapidly increased in blood of patients in septic shock and that lymphocyte apoptosis leads to a profound and persistent lymphopenia associated with poor outcome. These results suggest that lymphocyte apoptosis is one of the main components of human septic shock immune dysfunction and could be related more to microcirculatory disturbance than to circulating factors.

Fetal alcohol syndrome (FAS) is a common identifiable teratogenic cause of mental retardation, neurological deficit, mental disorders, and developmental disabilities. Accurate estimates of the cost of care for persons with FAS are essential for appropriate funding of health care, developmental disabilities services, special education, and other service systems, as well as prioritizing funding of public health prevention efforts. The cost of care for individuals with FAS can be conceptualized as the annual cost of care for one person or a population, or as the lifetime cost of care for an individual. Annual cost estimates for the United States range from $75 million in 1984 to $4.0 billion in 1998. Estimates of lifetime cost vary from $596,000 in 1980 to $1.4 million in 1988. After adjustments for changes in inflation and population, 2002 estimates of total annual cost and lifetime cost are higher. FAS is increasingly being recognized as a large public health problem with high potential for the prevention of future cases and for the prevention of excess disability and premature mortality in persons who are affected. Each day, from 6-22 infants with FAS are born in the United States, and as many as 87-103 more are born with other impairments resulting from prenatal alcohol exposure. Updated and improved cost data on FAS should be a research priority.

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)-mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-alpha or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-alpha is a potential target for cancer intervention.

Greater loss in structural integrity of the ipsilesional corticospinal tract (CST) is associated with poorer motor outcome in patients with hemiparetic stroke. Animal models of stroke have demonstrated that structural remodeling of white matter in the ipsilesional and contralesional hemispheres is associated with improved motor recovery. Accordingly, motor recovery in patients with stroke may relate to the relative strength of CST degeneration and remodeling. This study examined the relationship between microstructural status of brain white matter tracts, indexed by the fractional anisotropy (FA) metric derived from diffusion tensor imaging (DTI) data, and motor skill of the stroke-affected hand in patients with chronic stroke. Voxelwise analysis revealed that motor skill significantly and positively correlated with FA of the ipsilesional and contralesional CST in the patients. Additional voxelwise analyses showed that patients with poorer motor skill had reduced FA of bilateral CST compared to normal control subjects, whereas patients with better motor skill had elevated FA of bilateral CST compared to controls. These findings were confirmed using a DTI-tractography method applied to the CST in both hemispheres. The results of this study suggest that the level of motor skill recovery achieved in patients with hemiparetic stroke relates to microstructural status of the CST in both the ipsilesional and contralesional hemispheres, which may reflect the net effect of degeneration and remodeling of bilateral CST.

Studies of the biological effects of Fas signaling, using transformed cell lines as targets, indicate that ligation of the Fas receptor induces an apoptotic death signal. Chronically activated normal human T cells are also susceptible to Fas-mediated apoptosis. However, interactions between Fas and Fas ligand can also yield a costimulatory signal. Here, David Lynch, Fred Ramsdell and Mark Alderson present a model for the role of As and FasL in the homeostatic regulation of normal immune responses. They discuss how dysregulation of the Fas apoptotic pathway may contribute to certain disease states, including autoimmune disease and human immunodeficiency virus (HIV)-induced depletion of CD4+ T cells.

OBJECTIVE

Helicobacter pylori may affect the normal balance between gastric epithelial cell proliferation and epithelial cell death, thus interfering with the maintenance of gastric mucosal integrity. The aim of this study was to investigate the effect of H. pylori on cell growth, DNA synthesis, induction of apoptosis, and viability of human gastric epithelial cells in vitro.

METHODS

H. pylori was incubated with a differentiated human gastric cancer cell line for up to 72 hours, and the effects on cell numbers (cell counts and WST-1 assay), DNA synthesis (5-bromo-2'-deoxyuridine assay and [3H]thymidine incorporation), and DNA fragmentation (DNA fluorochrome staining, transmission electron microscopy, and histone enzyme-linked immunosorbent assay) were assessed.

RESULTS

Incubation of gastric epithelial cells with H. pylori led to a time- and concentration-dependent reduction of epithelial cell growth and a concomitant induction of DNA fragmentation. At high bacteria-cell ratios >> 100), inhibition of cell growth was associated with a reduction in DNA synthesis. Treatment of gastric cells with tumor necrosis factor alpha, a receptor-activating CD95/APO-1/Fas antibody, and interferon gamma markedly potentiated H. pylori-induced DNA fragmentation.

CONCLUSIONS

H. pylori affects gastric epithelial cell growth by direct induction of apoptosis and inhibition of DNA synthesis and indirectly by sensitization of epithelial cells for apoptosis induced by proinflammatory stimuli.

Focal ischemia by middle cerebral artery occlusion (MCAO) results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. Recent studies have shown activation of the extrinsic and intrinsic pathways of caspase-mediated cell death, as well as activation of the caspase-independent signaling pathway of apoptosis in several paradigms of focal cerebral ischemia by transient MCAO to adult rats and mice. The extrinsic pathway (cell-death receptor pathway) is initiated by activation of the Fas receptor after binding to the Fas ligand (Fas-L); increased Fas and Fas-L expression has been shown following focal ischemia. Moreover, focal ischemia is greatly reduced in mice expressing mutated (nonfunctional) Fas. Increased expression of caspase-1, -3, -8, and -9, and of cleaved caspase-8, has been observed in the penumbra. Activation of the intrinsic (mitochondrial) pathway following focal ischemia is triggered by Bax translocation to and competition with Bcl-2 and other members of the Bcl-2 family in the mitochondria membrane that is followed by cytochrome c release to the cytosol. Bcl-2 over-expression reduces infarct size. Cytochrome c binds to Apaf-1 and dATP and recruits and cleaves pro-caspase-9 in the apoptosome. Both caspase-8 and caspase-9 activate caspase-3, among other caspases, which in turn cleave several crucial substrates, including the DNA-repairing enzyme poly(ADP-ribose) polymerase (PARP), into fragments of 89 and 28 kDa. Inhibition of caspase-3 reduces the infarct size, further supporting caspase-3 activation following transient MCAO. In addition, caspase-8 cleaves Bid, the truncated form of which has the capacity to translocate to the mitochondria and induce cytochrome c release. The volume of brain infarct is greatly reduced in Bid-deficient mice, thus indicating activation of the mitochondrial pathway by cell-death receptors following focal ischemia. Recent studies have shown the mitochondrial release of other factors; Smac/DIABLO (Smac: second mitochondrial activator of caspases: DIABLO: direct IAP binding protein with low pI) binds to and neutralizes the effects of the X-linked inhibitor of apoptosis (XIAP). Finally, apoptosis-inducing factor (AIF) translocates to the mitochondria and the nucleus following focal ischemia and produces peripheral chromatin condensation and large-scale DNA strands, thus leading to the caspase-independent cell death pathway of apoptosis. Delineation of the pro-apoptotic and pro-survival signals in the penumbra may not only increase understanding of the process but also help to rationalize strategies geared to reducing brain damage targeted at the periphery of the infarct core.

Polyketide synthases (PKS) perform a stepwise biosynthesis of diverse carbon skeletons from simple activated carboxylic acid units. The products of the complex pathways possess a wide range of pharmaceutical properties, including antibiotic, antitumor, antifungal, and immunosuppressive activities. We have performed a comprehensive phylogenetic analysis of multimodular and iterative PKS of bacteria and fungi and of the distinct types of fatty acid synthases (FAS) from different groups of organisms based on the highly conserved ketoacyl synthase (KS) domains. Apart from enzymes that meet the classification standards we have included enzymes involved in the biosynthesis of mycolic acids, polyunsaturated fatty acids (PUFA), and glycolipids in bacteria. This study has revealed that PKS and FAS have passed through a long joint evolution process, in which modular PKS have a central position. They appear to have derived from bacterial FAS and primary iterative PKS and, in addition, share a common ancestor with animal FAS and secondary iterative PKS. Furthermore, we have carried out a phylogenomic analysis of all modular PKS that are encoded by the complete eubacterial genomes currently available in the database. The phylogenetic distribution of acyltransferase and KS domain sequences revealed that multiple gene duplications, gene losses, as well as horizontal gene transfer (HGT) have contributed to the evolution of PKS I in bacteria. The impact of these factors seems to vary considerably between the bacterial groups. Whereas in actinobacteria and cyanobacteria the majority of PKS I genes may have evolved from a common ancestor, several lines of evidence indicate that HGT has strongly contributed to the evolution of PKS I in proteobacteria. Discovery of new evolutionary links between PKS and FAS and between the different PKS pathways in bacteria may help us in understanding the selective advantage that has led to the evolution of multiple secondary metabolite biosyntheses within individual bacteria.

Calorie restriction (CR) increases longevity and retards the development of many chronic diseases, but the underlying metabolic signals are poorly understood. Increased fatty acid (FA) oxidation and reduced FA synthesis have been hypothesized to be important metabolic adaptations to CR. However, at metabolic steady state, FA oxidation must match FA intake plus synthesis; moreover, FA intake is low, not high, during CR. Therefore, it is not clear how FA dynamics are altered during CR. Accordingly, we measured food intake patterns, whole body fuel selection, endogenous FA synthesis, and gene expression in mice on CR. Within 2 days of CR being started, a shift to a cyclic, diurnal pattern of whole body FA metabolism occurred, with an initial phase of elevated endogenous FA synthesis [respiratory exchange ratio (RER) >1.10, lasting 4-6 h after food provision], followed by a prolonged phase of FA oxidation (RER = 0.70, lasting 18-20 h). CR mice oxidized four times as much fat per day as ad libitum (AL)-fed controls (367 +/- 19 vs. 97 +/- 14 mg/day, P < 0.001) despite reduced energy intake from fat. This increase in FA oxidation was balanced by a threefold increase in adipose tissue FA synthesis compared with AL. Expression of FA synthase and acetyl-CoA carboxylase mRNA were increased in adipose and liver in a time-dependent manner. We conclude that CR induces a surprising metabolic pattern characterized by periods of elevated FA synthesis alternating with periods of FA oxidation disproportionate to dietary FA intake. This pattern may have implications for oxidative damage and disease risk.

In vivo infection of lymphatic tissues by the human immunodeficiency virus type 1 (HIV-1) leads to enhanced apoptosis, which prominently involves uninfected bystander cells. Increased killing of such bystander cells is mediated in part through Nef induction of Fas ligand (FasL) expression on the surface of the virally infected T cells. The subsequent interaction of FasL with Fas (CD95) displayed on neighbouring cells, including HIV-1-specific cytotoxic T lymphocytes, may lead to bystander cell killing and thus forms an important mechanism of immune evasion. As HIV-1 also enhances Fas expression on virally infected cells, it is unclear how these hosts avoid rapid cell-autonomous apoptosis mediated through cis ligation of Fas by FasL. Here we show that HIV-1 Nef associates with and inhibits apoptosis signal-regulating kinase 1 (ASK1), a serine/threonine kinase that forms a common and key signalling intermediate in the Fas and tumour-necrosis factor-alpha (TNFalpha) death-signalling pathways. The interaction of Nef with ASK1 inhibits both Fas- and TNFalpha-mediated apoptosis, as well as the activation of the downstream c-Jun amino-terminal kinase. Our findings reveal a strategy by which HIV-1 Nef promotes the killing of bystander cells through the induction of FasL, while simultaneously protecting the HIV-1-infected host cell from these same pro-apoptotic signals through its interference with ASK1 function.

Intracellular fatty acid (FA) concentrations are in part determined by a regulated import/export system that is controlled by two key proteins, i.e. fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which respectively facilitate the transport of FAs across the cell membrane and their esterification to prevent their efflux. The aim of this investigation was to analyze the expression pattern of FATP and ACS and to determine whether their expression was altered by agents that affect FA metabolism through the activation of peroxisome proliferator-activated receptors (PPAR) such as the fibrates and thiazolidinediones. FATP mRNA was ubiquitously expressed, with highest levels being detected in adipose tissue, heart, brain, and testis. Fibrate treatment, which is known to preferentially activate PPARalpha, induced FATP mRNA levels in rat liver and intestine and induced ACS mRNA levels in liver and kidney. The antidiabetic thiazolidinedione BRL 49653, which is a high-affinity ligand for the adipocyte-specific PPARgamma form, caused a small induction of muscle but a robust induction of adipose tissue FATP mRNA levels. BRL 49653 did not affect liver FATP and had a tendency to decrease heart FATP mRNA levels. ACS mRNA levels in general showed a similar pattern after BRL 49653 as FATP except for the muscle where ACS mRNA was induced. This regulation of FATP and ACS expression by PPAR activators was shown to be at the transcriptional level and could also be reproduced in vitro in cell culture systems. In the hepatocyte cell lines AML-12 or Fa 32, fenofibric acid, but not BRL 49653, induced FATP and ACS mRNA levels, whereas in the 3T3-L1 preadipocyte cell line, the PPARgamma ligand induced FATP and ACS mRNA levels quicker than fenofibric acid. Inducibility of ACS and FATP mRNA by PPARalpha or gamma activators correlated with the tissue-specific distribution of the respective PPARs and was furthermore associated with a concomitant increase in FA uptake. Most interestingly, thiazolidinedione antidiabetic agents seem to favor adipocyte-specific FA uptake relative to muscle, perhaps underlying in part the beneficial effects of these agents on insulin-mediated glucose disposal.