ATP-binding cassette transporter A1 (ABCA1) is a cell membrane protein that exports excess cholesterol from cells to apolipoprotein (apo) A-I, the major protein in high density lipoproteins. Genetic studies have shown that ABCA1 protects against cardiovascular disease. The interaction of apoA-I with ABCA1 promotes cholesterol removal and activates signaling molecules, such as Janus kinase 2 (JAK2), that optimize the lipid export activity of ABCA1. Here we show that the ABCA1-mediated activation of JAK2 also activates STAT3, which is independent of the lipid transport function of ABCA1. ABCA1 contains two candidate STAT3 docking sites that are required for the apoA-I/ABCA1/JAK2 activation of STAT3. The interaction of apoA-I with ABCA1-expressing macrophages suppressed the ability of lysopolysaccaride to induce the inflammatory cytokines interleukin-1␤, interleukin-6, and tumor necrosis factor-␣, which was reversed by silencing STAT3 or ABCA1. Thus, the apoA-I/ABCA1 pathway in macrophages functions as an anti-inflammatory receptor through activation of JAK2/STAT3. These findings implicate ABCA1 as a direct molecular link between the cardioprotective effects of cholesterol export from arterial macrophages and suppressed inflammation.Two major processes that initiate the formation of atherosclerotic lesions in the artery wall are inflammation and the deposition of excess cholesterol in macrophages. It is believed that both of these events are in response to trapping of sterolrich lipoproteins in the artery, where they undergo oxidation and other modifications to become inflammatory stimuli that recruit and activate macrophages (1). These cells ingest and degrade the modified lipoproteins, leading to intracellular accumulation of cholesterol ester lipid droplets.Population studies have shown an inverse relationship between circulating levels of HDLs and risk for cardiovascular disease, implying that factors associated with HDL metabolism are cardioprotective. One of these factors is ABCA1, which exports cholesterol and phospholipids from cells to lipid-poor apoA-I to generate precursors for HDL particles (2). Because it is highly induced by sterols through nuclear receptors, ABCA1 is expressed in cholesterol-loaded cells, such as macrophages in atherosclerotic lesions (3). Loss-of-function mutations in ABCA1 accelerate atherosclerosis (4), which is likely to be the result of enhanced accumulation of cholesterolrich macrophages in arteries and the hyper-inflammatory responses of these cells.The cholesterol export function of ABCA1 occurs by a cascade of events involving direct binding of apoA-I to ABCA1, activation of signaling pathways, and solubilization of cholesterol and phospholipid domains formed by ABCA1 on the cell surface (5). We reported previously that incubating ABCA1-transfected baby hamster kidney (BHK) 2 cells with apoA-I or its synthetic mimetic peptides for only minutes dramatically increased autophosphorylation and thus activation of JAK2 by an ABCA1-dependent mechanism. Activation of JAK2 enhanced t...

ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation. Fig. S1]. In glycolysis, ATP is first invested in 2 phosphorylation reactions, catalyzed by hexokinase (HXK) and phosphofructokinase (PFK). Only further downstream in the pathway the ATP is regained and then a surplus of ATP is generated. In glycerol metabolism, ATP is invested in the reaction catalized by glycerol kinase (GK) (Fig. 1).In the absence of specific regulation of HXK and PFK, the ATP produced by glycolysis could boost the flux through these enzymes above the capacity of the enzymes downstream, and hexose phosphates [glucose 6-phosphate (Glc6P), fructose 6-phosphate (Fru6P) and fructose 1,6-phosphate (Fru1,6BP)] would accumulate to extreme levels. By analogy to the turbo engine (which uses engine exhaust to boost performance), this property was called the ''turbo-design''of glycolysis (2). Many organisms avoid the negative side effects of the autocatalytic design of glycolysis by a tight feedback regulation of HXK and PFK, e.g., inhibition of HXK by Glc6P (3) or trehalose 6-phosphate (5). In yeast, deletion of trehalose-6-phosphate synthase leads to glucose toxicity, accumulation of hexose phosphates and rapid consumption of ATP (4). This ''turbo-explosion'' phenotype is rescued by reducing the expression of HXK (5) or glucose transporters (6).Trypanosoma brucei, the tropical parasite that causes the deadly African sleeping sickness, lacks feedback regulation of HXK and PFK (7-9). The parasite has a complex life cycle that alternates between insect and mammalian hosts; in the latter it lives in the bloodstream, supplied with a ready source of glucose. How then are trypanosomes protected against a possible turboexplosion of glycolysis?A key feature of trypanosome glycolysis is the compartmentation of the first 7 enzymes of glycolysis and 2 involved in glycerol metabolism inside peroxisome-like organelles called glycosomes (10). In the glycosome, ATP and redox levels are ba...

Trypanosomatids, the etiologic agents of sleeping sickness, leishmaniasis, and Chagas' disease, compartmentalize glycolysis within glycosomes, metabolic organelles related to peroxisomes. Here, we identify a trypanosome homologue of PEX14, one of the components of the peroxisomal protein import docking complex. We have used double-stranded RNA interference to target the PEX14 transcript for degradation. Glycosomal matrix protein import was compromised, and both glycolytic bloodstream stage parasites and mitochondrially respiring procyclic stage parasites were killed. Thus, unlike peroxisomes, glycosomes are essential organelles. Surprisingly, procyclic forms, which can grow in the absence of glucose, were killed by PEX14 RNA interference only when simple sugars were present. Thus, interference with glycosome protein import makes glucose toxic to trypanosomes.

Ammonia switch-off is the immediate inactivation of nitrogen fixation that occurs when a superior nitrogen source is encountered. In certain bacteria switch-off occurs by reversible covalent ADP-ribosylation of the dinitrogenase reductase protein, NifH. Ammonia switch-off occurs in diazotrophic species of the methanogenic Archaea as well. We showed previously that in Methanococcus maripaludis switch-off requires at least one of two novel homologues of glnB, a family of genes whose products play a central role in nitrogen sensing and regulation in bacteria. The novel glnB homologues have recently been named nifI 1 and nifI 2 . Here we use in-frame deletions and genetic complementation analysis in M. maripaludis to show that the nifI 1 and nifI 2 genes are both required for switch-off. We could not detect ADP-ribosylation or any other covalent modification of dinitrogenase reductase during switch-off, suggesting that the mechanism differs from the well-studied bacterial system. Furthermore, switch-off did not affect nif gene transcription, nifH mRNA stability, or NifH protein stability. Nitrogenase activity resumed within a short time after ammonia was removed from a switched-off culture, suggesting that whatever the mechanism, it is reversible. We demonstrate the physiological importance of switch-off by showing that it allows growth to accelerate substantially when a diazotrophic culture is switched to ammonia.

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We designed a transposon insertion mutagenesis system for Methanococcus species and used it to make mutations in and around a nifH gene in Methanococcus maripaludis. The transposon Mudpur was constructed with a gene for puromycin resistance that is expressed and selectable in Methanococcus species. A 15.6-kb nifH region from M. maripaludis cloned in a vector was used as a target for mutagenesis. A series of 19 independent Mudpur insertions spanning the cloned region were produced. Four mutagenized clones in and around nifH were introduced by transformation into M. maripaludis, where each was found to replace wild-type genomic DNA with the corresponding transposon-mutagenized DNA. Wild-type M. maripaludis and a transformant containing a Mudpur insertion upstream of nifH grew on N 2 as a nitrogen source. Two transformants with insertions in nifH and one transformant with an insertion downstream of nifH did not grow on N 2 . The transposon insertion-gene replacement technique should be generally applicable in the methanococci for studying the effects of genetic manipulations in vivo.Genetic approaches to the study of methanogenic Archaea are becoming feasible because of the development of methods for genetic transformation and selection in Methanococcus species (3,6,10,14). In both Methanococcus voltae (6) and Methanococcus maripaludis (12), a puromycin resistance gene (6) can be introduced by transformation. The resistance gene is maintained after integration into the genome by recombination events that are facilitated by the presence of genomic fragments in the introduced DNA. These developments make it possible to produce mutations in cloned genes and to observe the effects in vivo. We have developed a transposon insertion mutagenesis technique that builds upon these advances and have used it to test the effects of mutations in and around a nifH gene of M. maripaludis. MATERIALS AND METHODSGrowth of bacteria. Strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were maintained at 30ЊC (MH132 and P2392) or 37ЊC (DH5␣F) in Luria broth (LB [9]) or NZY (0.5% NaCl, 0.2% MgSO 4 , 0.5% yeast extract, 1% Casamino Acids [pH 7.5]). Ampicillin and chloramphenicol were used at a concentration of 25 g/ml unless otherwise stated. The techniques used for growing methanogens were those of Balch et al. (2). M. maripaludis was grown at 30ЊC in medium number 3 (2) with the following modifications. Vitamins, sodium acetate, yeast extract, and Trypticase were omitted. The trace mineral solution was supplemented with NiCl 2 ⅐ 6H 2 O (0.025 g/liter), NaSeO 3 (0.2 g/liter), and Na 2 WO 4 ⅐ 2H 2 O (0.1 g/liter), and the amount of Na 3 MoO 4 ⅐ 2H 2 O was increased to 0.1 g/liter as described in reference 15. For maintenance of M. maripaludis strains containing Mudpur, puromycin was added to 2.5 g/ml. Nitrogen-free medium was further modified so that all forms of combined nitrogen were lacking. Fe(NH 4 ) 2 (SO 4 ) 2 was replaced by FeSO 4 ⅐ 7H 2 O (0.01 g/liter), and NH 4 Cl and cysteine were omitted. I...

We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 M, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases.Nitrogen fixation, the conversion of atmospheric N 2 to ammonia, is carried out by a variety of Bacteria and Archaea (23). Among the Bacteria, it occurs in at least four groups, the Proteobacteria, gram-positive bacteria, cyanobacteria, and green sulfur bacteria. Among Archaea, nitrogen fixation is found in all three orders of methanogens (10). Despite its wide phylogenetic distribution, the mechanism of nitrogen fixation is conserved. A typical dinitrogenase is an ␣ 2 ␤ 2 tetramer encoded by nifD and nifK and containing an iron-molybdenum cofactor, FeMoCo (22). Mo-independent dinitrogenases (2) have cofactors that coordinate vanadium in place of molybdenum (Vdinitrogenases) or that have neither molybdenum nor vanadium (Fe-dinitrogenases); these are encoded by the nifD and nifK homologs vnfD and vnfK and anfD and anfK, respectively. An additional small subunit (␦) found with the Mo-independent dinitrogenases is encoded by vnfG or anfG. Included in all nitrogenase complexes is dinitrogenase reductase, typically a homodimer in Bacteria, encoded by nifH, vnfH, or anfH.The discovery of nitrogen fixation in methanogens in 1984 redefined the phylogenetic extremity of nitrogen fixation and opened the door to a series of new questions regarding biological nitrogen fixation (1, 12). Many of the properties common to nitrogen fixation in the Bacteria hold true in the Archaea as well. Lobo and Zinder found the nitrogenase complex from Methanosarcina barkeri to resemble those from Bacteria in subunit composition (except that dinitrogenase reductase appeared to be a homotetramer), substrate range, and immunological cross-reactivity of dinitrogenase reductase (9). nif genes from a variety of methanogens proved to be homologous and similar in organization to those from Bacteria (10). Phylogenetic analysis of nifH, nifD, and nifK showed the methanogen genes to be related to certain groups of bacterial genes (discussed below).One question that has not been resolved is which metals are required for nitrogen fixation in methanogens and might therefore be incorporated into the dinitrogenase cofactor. Lobo and Zinder found that molybdenum but not vanadium stimulated nitrogen fixation in M. barkeri 227 (8). In contrast, Scherer reported stimulation by molybdenum and to a lesser extent by vanadium in two strains of M. barkeri, 227 and Fusaro (14). The issue is further complicated by the fact that M. barkeri 227 has two potentially active nitrogenases, as evidenced by the existence of two sets o...

Thirty-six patients with Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph+ ALL) were studied for the presence of the bcr-abl fusion mRNA transcript after an allogeneic matched related (N = 12), partially matched related (N = 4), matched unrelated (N = 14), autologous (N = 5), or syngeneic (N = 1) bone marrow transplant (BMT). Seventeen were transplanted in relapse, and 19 were transplanted in remission. Twenty-three patients had at least one positive bcr-abl polymerase chain reaction (PCR) assay after BMT either before a relapse or without subsequent relapse. Ten of these 23 relapsed after a positive assay at a median time from first positive PCR assay of 94 days (range, 28 to 416 days). By comparison, only 2 relapses occurred in the 13 patients with no prior positive PCR assays; both patients had missed at least one scheduled follow-up assay and were not tested 2 months and 26 months before their relapse. The unadjusted relative risk (RR) of relapse associated with a positive PCR assay compared with a negative assay was 5.7 (95% confidence interval 1.2 to 26.0, P = .025). In addition, the data suggest that the type of bcr-abl chimeric mRNA detected posttransplant was associated with the risk of relapse: 7 of 10 patients expressing the p190 bcr-abl relapsed, compared with 1 of 8 who expressed only the p210 bcr-abl mRNA (P = .02, log-rank test). The RR of p190 bcr-abl positivity compared to PCR-negative patients was 11.2 (confidence interval 2.3-54.8, P = 0.003), whereas a positive test for p210 bcr-abl was apparently not associated with an increased relative risk. In separate multivariable models, PCR positivity remained a statistically significant risk factor for relapse after separately adjusting for donor (unrelated and partially matched v matched, autologous, and syngeneic), remission status at the time of transplant, the presence of acute graft-versus-host disease (GVHD), and type of conditioning regimen (total body irradiation dose of ≤1,200 cGy v <1,200 cGy). The PCR assay appears to be a useful test for predicting patients at high risk of relapse after BMT and may identify patients who might benefit from therapeutic interventions. The finding that the expression of p190 bcr-abl may portend an especially high risk of relapse suggests a different clinical and biologic behavior between p190 and p210 bcr-abl.