Thus, activation requires conformational changes across the interface that coordinate the positioning of catalytic residues in both GTPase sites. A distinct class of mutants exhibits half-site reactivity and thus allows us to further uncouple the activation of individual GTPases. Our dissection of the activation process suggests discrete conformational stages during formation of the active SRP•SRP receptor complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.The signal recognition particle (SRP) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane. During this process, two GTPases, one in SRP and one in the SRP receptor , form a complex in which both proteins reciprocally activate the GTPase reaction of one another. Here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the Ffh-FtsY interaction. Mutations of a large number of residues at the interface impair complex formation, supporting the importance of an extensive interaction surface. Surprisingly, even after a stable complex is formed, single mutations in FtsY can block the activation of GTP hydrolysis in  A molecular dissection of the bacterial signal recognition particle-receptor complex reveals discrete stages that provide control points in the protein targeting reaction, ensuring appropriate timing of binding and release GTPases comprise a superfamily of proteins that provide molecular switches to regulate many cellular processes, including translation, signal transduction, cytoskeletal organization, vesicle transport, nuclear transport, and spindle assembly . In manyTwo homologous GTPases, one in the signal recognition particle (SRP) and one in the SRP receptor , mediate the cotranslational targeting of membrane and secretory proteins to the eukaryotic endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. During the targeting reaction, SRP and SR switch between different functional states . SRP firHowever, the regulatory mechanism of the SRP family GTPases provides a notable exception to the “GTPase switch” paradigm. Unlike many other GTPases, no external GEFs or GAPs are known for the SRP and SR GTPases. Instead, Ffh and FtsY bind nucleotides weakly, and nucleotide dissociation and exchange are very fast ; thus, tRas GTPase fold. In addition, all SRP family GTPases contain a unique “N” domain, which together with the G domain forms a structural and functional unit called the NG domain. The crystal structures of the individual Ffh and FtsY NG domains show that both proteins have a wide-open GTP-binding pocket, and the apoforms of these proteins are stabilized by a network of side-chain interactions in the empty active site  How do these GTPases act as reciprocal activating proteins for one another, and (ii) how does the SRP family of GTPases switch between the “on” and “off” states, as the GTPases are predominantly in the GTP-bound state as they enter the targeting cycle and no stable, GDP-bound state exists under cellular conditions?Escherichia coli FtsY and showed that mutations that have deleterious effects on the Ffh-FtsY interaction define a large surface patch on FtsY that lies on its interaction surface with Ffh identified in the crystal structure  does not equal the dK (16 nM) of the Ffh•FtsY complex. Likewise, the mK of the reaction with mutant FtsY does not necessarily equal the dK of the mutant Ffh•FtsY complex, and thus cannot meaningfully distinguish between mutational effects on complex formation and GTPase activation.In contrast, for Class II and III mutants, the rate of GTPase reaction remains slow even at saturating concentrations of FtsY (data for representative mutants are shown in 0k). Addition of mutant FtsY [FtsY(mt)], which can form a complex with Ffh, will sequester the Ffh molecules into a less active Ffh•FtsY(mt) complex  complex.To circumvent these problems, we devised an assay to determine the ability of each FtsY mutant to inhibit the interaction of wild-type FtsY with Ffh. This assay allowed us to monitor selectively complex formation between Ffh and the FtsY mutants. The conditions of the assay were designed so that in the absence of any mutant FtsY as an inhibitor, a robust GTPase reaction mediated by Ffh and wild-type FtsY was observed A, k0. Ad2-fold weaker than the affinity of wild-type FtsY for Ffh  stood out as strong inhibitors. These mutants -specific Ffh mutant, Ffh D251(248)N. Asp251(248), located in the GTP-binding consensus motif, is conserved throughout the GTPase superfamily and forms a hydrogen bonding network with the N2 and N3 amino protons on the guanine ring . In the presence of XTP, Ffh D251(248)N stimulates the GTPase reaction of FtsY and, reciprocally, its XTPase reaction is stimulated by FtsY in the presence of GTP. We therefore used Ffh D251(248)N to monitor the individual hydrolysis events—XTP hydrolysis from Ffh D251(248)N and GTP hydrolysis from the mutant FtsY constructs—in the Ffh D251(248)N•FtsY complex.The mutants described above were identified by analyzing the sum of the two GTP hydrolysis reactions from both Ffh and FtsY. All of the Class II and III mutants must be defective in ine ring . The AspAs expected, all of the Class II and Class III mutants were defective in both hydrolysis reactions and, similarly, all but one Class I mutant and most of the neutral mutants showed no significant defect in either of the two reactions (unpublished data). Five half-site mutants, however, stood out from the pool of originally categorized Class I and neutral mutants see , green. 2-fold, whereas the reciprocal reaction, GTP hydrolysis by Ffh, is reduced only 2- to 4-fold N, thereby reversing the nucleotide specificity of the two binding partners. Upon complex formation, FtsY D449(248)N becomes XTP specific and reciprocally activates GTP hydrolysis in Ffh. Consistent with the results observed with Ffh D251(248)N, all Class IV mutations thus analyzed reduce the rate of XTP hydrolysis from mutant FtsYs by more than 10o 4-fold . Thus taThe mutational analyses described here define four distinct classes of mutants that map to the Ffh-FtsY interface. Each mutant class blocks the reaction in a different way and at a distinct stage, demonstrating that (i) multiple conformational rearrangements are required to form an activated Ffh•FtsY complex and (ii) some rearrangements can be blocked without preventing other rearrangements from taking place. The different classes of mutant interrupt the reaction in different ways, as represented by the states depicted in We previously showed that FtsY exhibits little discrimination between different nucleotides in its free, uncomplexed form, but gains substantial specificity for GTP only in the Ffh•FtsY complex . We therThe crystal structure of the Ffh•FtsY complex also shows that Ffh undergoes similar N-G domain rearrangements upon complex formation, although the effects of this rearrangement on nucleotide specificity are less apparent, as free Ffh already displays significant discrimination between nucleotides . Indeed, mutations at the N-G domain interface in either Ffh or FtsY impair complex formation, supporting the functional importance of this rearrangement in both binding partners . We propboth active sites, the defect in these mutants is not a consequence of simply removing a catalytic residue. Rather, this suggests that even after a stable, closed complex is formed, activation requires additional conformational changes (the “docking” process) that align active-site residues with respect to the bound nucleotides in both GTPase sites , suggesting that the mutant proteins have assumed the closed conformation in the complex. Because single mutations in FtsY can disrupt GTPase activation in The model in 135TFRAAA). As concluded from the structure, this loop can move relatively independently from the rest of the protein  comes from three of the Class II mutants, , which all map to the conserved IBD loop  to either Ala or Trp also results in a Class II phenotype. This residue in FtsY hydrogen bonds across the interface to the ribose 3′-OH of the nucleotide bound to Ffh. The ribose 3′-OH reciprocally donates a hydrogen bond back to the γ-phosphate of the twinned substrate in FtsY  to inhibit the interaction between Ffh and wild-type FtsY, as described in detail in the text see A. The da2+ as described in Fluorescence emission spectra were acquired as described in


Statins also inhibit the isoprenoid pathway, thereby modulating the activities of the Rho family of small GTPases—Rho A, B, and C—as well as the activities of Rac and cdc42. Rho proteins, in turn, exert many of their effects via Rho-associated protein kinases (ROCKs). Several cell-surface molecules are substrates for activated α-secretase-type ectodomain shedding, and regulation of shedding typically occurs via activation of protein kinase C or extracellular-signal-regulated protein kinases, or via inactivation of protein phosphatase 1 or 2A. However, the possibility that these enzymes play a role in statin-stimulated shedding has been excluded, leading us to investigate whether the Rho/ROCK1 protein phosphorylation pathway might be involved.Statins are widely used cholesterol-lowering drugs that act by inhibiting HMGCoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent evidence suggests that statin use may be associated with a decreased risk for Alzheimer disease, although the mechanisms underlying this apparent risk reduction are poorly understood. One popular hypothesis for statin action is related to the drugs' ability to activate α-secretase-type shedding of the α-secretase-cleaved soluble Alzheimer amyloid precursor protein ectodomain (sAPPα shedding from a neuroblastoma cell line via a subcellular mechanism apparently located upstream of endocytosis. A farnesyl transferase inhibitor also increased sAPPα shedding, as did a dominant negative form of ROCK1. Most conclusively, a constitutively active ROCK1 molecule inhibited statin-stimulated sAPPα shedding.We found that both atorvastatin and simvastatin stimulated sAPPα from cultured cells, at least in part, by modulation of the isoprenoid pathway and ROCK1.Together, these data suggest that statins exert their effects on shedding of sAPP The cholesterol-lowering statins seem to have a protective effect against Alzheimer disease. In cultured cells, statins affect cleavage of the amyloid precursor protein via the ROCK kinase. This could explain some of their beneficial effects Typical maturation and half-life of mature cellular holoAPP were observed, as was subsequent release of sAPPα [t = 0 versus a control [vehicle treatment] of 150% of the level of immature APP at t = 0; also, at t = 30 min, the relative percent values for drug versus vehicle were 380% and 200%, respectively). Fold increases in released sAPPα in the same experiments were approximately 3- to 4-fold . Secretory maturation toxicity is one possible mechanism for elevated levels of intracellular mature holoAPP and causes retarded conversion of mature holoAPP to sAPPα. This pattern was not observed following statin treatment, excluding maturation toxicity as a mechanism underlying the altered levels of mature cellular holoAPP.A pulse-chase protocol was also used to study post-transcriptional regulation of APP metabolsm by statins (data not shown). This protocol avoids any confound that might arise because of altered APP transcription. Pulse-chase studies were performed using a 10-min pulse with 3Smethioniof sAPPα . In the α. However, the fold effect on reduced intracellular turnover in the endosomal/lysosomal pathway  is apparently insufficient to explain the fold effect on sAPPα generation , indicating a contribution from a downstream site in the processing pathway. When these results are taken together with independent work on regulated shedding of transforming growth factor α (TGFα) [Instead, the pattern that we observed raises the possibility that statins, presumably via isoprenoids , as discussed in the next section, may alter sorting of cellular holoAPP, diverting holoproteins away from terminal degradation in the endosomal/lysosomal pathway and into the constitutive secretory pathway that generates sAPPα (TGFα) ,36, a paα or TGFα [The identification of the regulatory components of the ectodomain shedding machinery have been long-sought in other studies employing phorbol esters to stimulate shedding of sAPP or TGFα ,35,36,37 or TGFα . In our NPXY clathrin-coated vesicle targeting sequence [α shedding, presumably by extending the half-life of co-localized α-secretase and APP on the plasma membrane. In order to exclude the possible contribution of altered endocytosis to statin-stimulated shedding, we evaluated the effect of phenylarsine oxide (PO), an inhibitor of endocytosis, on statin-stimulated shedding . The additivity of statin- and PO-stimulated shedding is consistent with the hypothesis that statins act at or near the plasma membrane, prior to internalization of csAPP. Under all circumstances, stimulated sAPPα shedding was completely blocked using TNFα protease inhibitor 2, a standard α-secretase/metalloproteinase inhibitor . In the same experiment, levels of holoAPP were modestly increased but, again, to an extent insufficient to account for the increase in shed sAPPα  of mevalonic acid did not affect statin-induced sAPPα shedding, but complete inhibition of statin-activated sAPPα shedding was achieved with higher doses of mevalonic acid (100 μM). As predicted, the shedding observed following treatment with FTI-1 was not inhibited by any of the concentrations of mevalonic acid tested . Conversely, DN ROCK1 alone activated shedding of sAPPα. Cellular levels of holoAPP were not affected by transfection (p < 0.05 versus GFP control). In studies aimed at independent confirmation of the involvement of ROCK activation in sAPPα shedding, we treated SweAPP N2a cells with arachidonic acid, an activator of ROCK. As shown in α without altering levels of holoAPP. Based on this series of results, we concluded that both basal and activated sAPPα shedding from cultured cells are controlled by ROCK activity.Since many isoprenoid-mediated Rho effects converge on ROCKs, we next transfected N2a cells with cDNAs encoding either green fluorescent protein (GFP) (control), CA ROCK1, or DN ROCK1 . Simvastα release and blocked statin-activated sAPPα shedding (data not shown). This result was unexpected in light of the effects of DN ROCK1. Given the internally consistent actions of DN ROCK1 and CA ROCK1, as well as the results employing either FTI-1 or arachidonate, we concluded that the Y-27632 result might be due to inhibition by Y-27632 of protein kinases other than ROCK1 [In some experiments, cells were treated with Y-27632 (10 nM to 50 μM), a compound that can inhibit ROCKs. Y-27632 showed no effect on basal sAPPan ROCK1 . The posThe isoprenoid pathway involves lipid modification of various members of the Rho family of small GTPases by the addition of either farnesyl or geranylgeranyl moieties ,42. Isopα in the cerebrospinal fluid and blood. This aspect of APP metabolism bears resemblance to the proteolytic signal transduction pathways involved in processing pro-TGFα [The first evidence that APP might be a substrate for ectodomain shedding was provided by Weidemann et al.  who idenpro-TGFα ,36 and Npro-TGFα . In the pro-TGFα ,29,45. Ipro-TGFα . Ultimatpro-TGFα .α rises from 2 mol shed per 10 mol synthesized to 4 mol shed per mole synthesized. Most of this shedding is catalyzed by the α-secretase pathway, but a trace amount  is cataunt  or by terminating intercellular signaling . A popular model holds that sAPPα may function as a neurotrophic and/or neuroprotective factor, and may promote neurite outgrowth [What is the function of shed sAPPutgrowth . More reutgrowth . Targeteutgrowth , presumautgrowth . Mice wiCao and Sudhof  have recThe potential role of cholesterol in α-secretase-mediated shedding was discovered by Bodovitz and Klein  who usedα following statin application.Maillet et al.  implicatα shedding, i.e., activation of ROCK1 blocks basal and stimulated shedding while ROCK1 inhibition apparently relieves a tonic negative influence exerted on shedding by ROCK1 activity.CA ROCK1 and DN ROCK1 molecules yielded direct and complementary evidence that ROCK1 was indeed a candidate for modulation of statin-activated α-secretase action. Further, we were able to demonstrate that α-secretase activity could be modulated by molecules further upstream in the isoprenoid pathway see . FTI-1, As in PKC- and ERK-activated shedding, the ROCK1 substrate effector molecule or molecules that regulate proteolysis by ADAMs remain to be identified. The cytoplasmic domains of both APP and ADAM-17 have been evaluated as candidates for important targets of protein phosphorylation during the regulated shedding process, but neither “substrate activation” nor “enzyme activation” appears to explain the phenomenon, i.e., phosphorylation of neither APP nor ADAM-17 dramatically increases the efficiency of α-secretion ,62, indiOur data using statins and PO localize the mediator of statin-activated shedding to the plasma membrane, upstream of endocytosis, as appears to be the case for PKC-activated shedding ,63,64,65α-Secretase activation is a potential therapeutic strategy for modifying cerebral amyloidosis in Alzheimer disease . This prPreliminary results from a pilot proof-of-concept using atorvastatin in a human clinical treatment trial are consistent with the proposed beneficial effects of this class of compounds . Since aThe results reported here point to several areas for additional investigation. As described above, the key substrate or substrates linking cytoplasmic protein phosphorylation to intralumenal or cell-surface protelysis remain to be identified. Nonetheless, α-secretase activation has been validated as a viable therapeutic strategy for modulating cerebral amyloidosis . Identifhttp://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the proteins discussed in this paper are ROCK1 (NP_005397) and APP695, 595–596 KM/NL (NP_958817).The GenBank  is broken down (or cleaved). There are two ways in which APP can be broken down. “Bad cleavage” releases the toxic amyloid-beta, whereas “good cleavage” destroys it. When researchers gave statins to animals over a long period of time, they found that statins could slow down the formation of amyloid plaques. From the animal experiments, it seemed that statins somehow caused more good cleavage to occur.This study examined how statins can affect APP cleavage.They studied cells to see which of the players known to be involved in APP cleavage were affected by statin.Statin's ability to promote “good cleavage” of APP involves a molecular pathway called the Rho/ROCK1 pathway. It seems that when ROCK1 is active, less good cleavage takes place. But in the presence of statins, ROCK1 is less active, shifting the balance toward good cleavage. Consistent with this, when the scientists blocked the Rho/ROCK1 pathway, they saw the good cleavage pattern even without statin.Inhibition of the Rho/ROCK1 pathway could explain some of the beneficial effects of statins against Alzheimer disease. And the pathway itself seems worth more research to see whether it might be a good target for new ways to prevent and treat Alzheimer disease.Statins are likely to influence the risk for Alzheimer disease by several different pathways, and future studies will need to show how important this particular pathway is in the overall picture. Moreover, studies like this one are by necessity done in cells under carefully controlled laboratory conditions and still a long way from the development of safe and effective drugs.http://www.alz.org/Resources/TopicIndex/statins.aspFactsheet on statins from the Alzheimer's Association: http://www.alzheimers.org/index.htmlGeneral information at the Alzheimer's Disease Education and Referral Center at the United States National Institute of Aging: http://www.alz.co.uk/Homepage of Alzheimer's Disease International, an umbrella organization of Alzheimer disease associations around the world:


The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP·SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation. The functional core proteins of the signal recognition particle (SRP) and the SRP receptor (SR)  contain GTPase domains and form a distinct subfamily of GTPases. These GTPases mediate cotranslational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER) membrane in eukaryotes or the plasma membrane in prokaryotes  A family of crystal structures of prokaryotic SRP GTPases illustrates this unique mechanism of activation. In particular, apo structures of Ffh-NG, a truncated version of the prokaryotic SRP core protein containing the amino- and GTPase domains, and FtsY, the SR protein, show the stabilization of an “open” state through interactions of the GTPase and SRP conserved sequence motif residues 4(i) the structures observed represent a ground state of the GTP hydrolysis reaction, and (ii) that additional conformational changes in the active site are necessary to progress to the transition state. Recently, mutational studies of FtsY have revealed that site-specific mutations can modulate discrete conformational changes during Ffh·FtsY complex formation The structures of the FtsY·Ffh-NG complex in presence of the non-hydrolyzable substrate analog GMPPCP Here we report the structures of two conformations of FtsY in complex with the substrate analogue GMPPNP. These structures reveal two novel active site architectures for SR GTPase-nucleotide complexes. These structures, along with the structure of apo FtsY and FtsY in the Ffh-NG·FtsY complex, can be interpreted as a series of discrete conformational states along the pathway of step-wise activation of the SRP GTPases during the formation of the SRP·SR complex and provide structural explanations for the biochemical differences observed for FtsY along the targeting cycle.Thermus aquaticus (T. aquaticus) were crystallized and structures determined. Crystal form one initially grew out of a purified complex of FtsY, full length Ffh from T. aquaticus, GMPPNP and Mg2+. Subsequent crystals were grown in the absence of Ffh. The structure of this form was determined to 2.2 Å resolution and contained a single non-crystallographic (NCS) two-fold related dimer per asymmetric unit with one monomer in apo-FtsY and the other FtsY monomer bound with GMPPNP  is involved in maintaining the active site cavity spacing through interactions with the P-loop and an aspartate in the α3 helix , a unique motif II sequence element contains two critical residues: Arg142 and Gln148. In complex with Ffh-NG, FtsY residue R142 interacts with γ-phosphate and Gln148 helps coordinates the magnesium ion . A strikBinding of nucleotide is expected to confer certain conformational changes in the protein to both accommodate the nucleotide and to orient the active site moieties for proper hydrolysis. However, as also observed in Ffh-NG bound with GMPPNP, FtsY does not appear to be in a state that would allow for proper nucleotide hydrolysis. Lys115 is displaced from the active site cavity and does not interact with the phosphate groups of GMPPNP . Also abThe structures of FtsY described here represent snapshots of FtsY and nucleotide in various active site conformations. We interpret these structures as representing discrete states along the pathway of activation and dissociation of FtsY in the targeting cycle. These structures can also be compared with the product complex of FtsY with GDP bound −3-fold The most obvious result in our study relates the variation in the position of conserved motif IV with a structural explanation for the poor base specificity and promiscuous nucleotide hydrolysis reported in nucleotide binding studies of FtsY by Shan and Walter trans substrate of Ffh-NG, a hydrogen bond with the 3′O of the GMPPCP. Analogous conserved residues act in the same manner in the Ffh-NG component of the complex. Further, the nucleotides themselves act in trans to stabilize the negative change on the γ-phosphate formed during GTP hydrolysis in contrast to most other GTPases that require an external activation residue from their respective GAPs. Both F1 and F2 show a total disruption in these interactions and in fact exhibit a coordination that positions the γ-phosphate out of the active site. Without these intra- and interactions, hydrolysis of GTP is not favored.The conserved structural elements in F1 and further in F2 suggest a sequential ordering of the active site during complex formation. The increasing coordination between Asp258 and GMPPNP from apo FtsY to F2 and further in F1 relates to a closing of the active site and an increase in the coordination of the guanine base of the nucleotide. Complex formation with Ffh-NG directs FtsY IBD residues Arg142 and Gln148 to interact with the β- and γ-phosphate groups while catalytic Asp139 activates the proposed nucleophilic water. In addition, Asn111 forms the only sidechain contact with the T.aquaticus FtsY lacks the A-domain, but retains an N-terminal helix, termed N1. The N1 helix becomes susceptible to proteolysis upon formation of targeting complex with Ffh T. aquaticus binds GDP in a canonical manner including the specificity-determining hydrogen bonding interactions with Asp258 i) the proteolytic susceptibility of N1 upon complex formation, (ii) increase in GTPase activity upon deletion of N1 and (iii) the observation of a canonical active site for monomeric FtsY:GDP, the N1 helix might act to repress GTPase activity in the monomeric form of FtsY. This could be accomplished by favoring an N/G domain organization that favors a more open active site. Upon complex formation, N1 rearranges for proper complex formation and active site optimization. In this manner, the N1 helix would act as a negative regulatory element; maintaining low activity in the non-targeting, monomeric form of FtsY as observed in the structures presented in this paper. This regulation might also be affected by interactions with the membrane and membrane-bound translocon.The A-domain is an N-terminal domain which has been proposed to anchor FtsY to the membrane 2+ coordination, perhaps by displacement of Mg2+ from the active site 2+ and in turn contributes to the low affinity for nucleotide through disruption of proper coordination architecture in the monomer form. The N1 helix might also contribute to regulation of the GTPase cycle and promote a step-wise activation by repressing GTPase activity in the monomeric form. The FtsY GMPPNP structures described here depict conformational states along the activation pathway for SRP GTPases in the targeting reaction and reveal the structural basis for the futile nature of GTP hydrolysis in monomeric FtsY. In addition, these structures provide insight into the step-wise ability of FtsY to self-regulate its GTPase activity as well as reset to the apo state through release of the nucleotide for subsequent rounds of targeting.GTP-hydrolysis by GTPases is intrinsically slow but can be accelerated by binding of specific external factors. SRP GTPases are unique in that activation of GTP-hydrolysis occurs upon formation of a complex of two GTPases. Our data support and expand an emerging model that explains some of the complexity in the catalytic cycle of FtsY and Ffh. SRP GTPases are regulated not at the point of nucleotide binding or exchange, but rather through a ‘step-wise’ mechanism. FtsY and Ffh bind GTP in an “open” or “primed” state. An “active” conformational state is reached upon the concerted rearrangements of the SRP GTPase conserved sequence elements after complex formation with SRP. A concerted step-wise formation would also imply that nucleotide hydrolysis and release might also be step-wise. Nucleotide release in GTPases is typically regulated by guanine nucleotide exchange factors by disruption of MgT. aquaticus FtsY was expressed in E. coli strain BL21 (DE3) (Novagen) using a pET21 plasmid and purified to homogeneity. FtsY was concentrated to 9 mg ml−1 in 50 mM Hepes pH 7.5, 150 mM potassium acetate pH 7.5, 2.5mM magnesium acetate and 2 mM β-OG, octyl-β-d-glucoside. Ffh was prepared as previously described 2 were carried out as hanging-drops with the vapor diffusion method at room temperature and crystals initially grew out of two conditions: 2.0 M ammonium sulfate and 2.0 M ammonium sulfate, 100 mM sodium acetate. Identification of FtsY as the sole component of the crystals was evaluated by gel electrophoresis and mass spectrometry. Additional crystals were grown from 18% PEG 2000, Tris pH 7.5, 100mM NaCl . For MAD, a mercury derivative of FtsY was used. Purified FtsY was incubated with 10 mM TCEP-HCl (Tris(2-Carboxyethyl) Phosphine, Hydrochloride) for 4 hours to reduce oxidation on the cysteine (Cys136). Following buffer exchange with a Hepes buffer to eliminate reducing agent, 10mM of the Hg compound CMNP  was added to the protein and incubated for 4 hours. The FtsY-Hg complex was then buffer exchanged with Hepes buffer using Centricon YM-10K (Amicon) to eliminate unbound Hg. The covalent binding of Hg was confirmed with mass spectrometry.f″ = 1.006 Å, λf′ = 1.009 Å and λhigh remote = 0.993 Å. Data collected at SSRL were integrated and reduced with MOSFLM and SCALA Data collection parameters and statistics for all data are listed in E. coli FtsY structure into the electron density. Initial density in the active site from the MAD phased maps as well as simulated annealing omit maps calculated from the molecular replacement model showed clear density for the tri-phosphate nucleotide. Figures were generated using SPDBV and PyMol.Two mercury sites were resolved by hand and confirmed using an automated heavy atom search procedure in CNS


TNF) and lymphotoxin-α  have been associated with asthma and atopy in some studies. Parental smoking is a consistent risk factor for childhood asthma. Secondhand smoke and ozone both stimulate TNF production.Polymorphisms in the proinflammatory cytokine genes tumor necrosis factor-α  in TNF-308 SNP was associated with increased risk of asthma , especially among children of non-smoking parents . Similarly, the A allele of the TNF-238 SNP was associated with increased asthma risk among children of nonsmoking parents . LTA SNPs were not associated with asthma. Haplotype analyses reflected the single SNP findings in magnitude and direction. TNF and LTA SNPs were not associated with the degree of atopy.The A allele of the TNF may contribute to childhood asthma and that associations may be modified by parental smoking.Our results suggest that genetic variation in  TNF [GenBank accession no. X02910 ] and LTA [GenBank accession no. X01393 ] genes are located consecutively in the class III region of the human major histocompatibility complex (MHC) on chromosome 6p21, which has shown evidence of linkage to asthma, atopy, and related phenotypes in multiple genome-wide studies , the defining member of the TNF family of cytokines, has been directly implicated in asthmatic airway inflammation and bronchial hyperresponsiveness . Lymphot studies .TNF and LTA influence gene expression. (TNF promoter region (TNF-308) and a SNP within the first intron of LTA (LTA NcoI) affect the rate of gene transcription and protein production  of both ression. . In partoduction . Severaltibility , althougtibility . The LTAh asthma ; howeverh asthma . The TNFnd atopy . The effTNF has been identified as a candidate gene for ozone-induced airway inflammation and hyperresponsiveness . Children and parents provided blood samples as sources of DNA. A parent, nearly always the mother, completed a questionnaire on the child’s symptoms and risk factors for asthma including current parental smoking, parental smoking during the first 2 years of the child’s life, maternal smoking during pregnancy, and residential history.We used the case–parent triad design . The stuhttp://www.ine.gob.mx/dgicurg/calaire/tend/concentra.php). Ozone levels were measured via ultraviolet photometry . The residence of each child who participated in this study was located using a map, and the closest monitoring station was assigned to that residence . Parents provided the written informed consent for the child’s participation. Children also gave their informed assent.1)  . ChildreAspergillus fumigatus, Alternaria, Mucor, Blattella germanica, Periplaneta americana, Penicillium, cat, dog, horse, Dermatophagoides (pteronyssinus and farina), Ambrosia, Artemisa ludoviciana, Cynodon dactylon, Chenopodium album, Quercus, Fraxinus, Helianthus, Ligustrum vulgare, Lolium perenne, Plantago lanceolata, Rumex crispus, Schinus molle, Salsola, and Phleum pratense. Histamine was used as a positive control and glycerin as a negative control. Children were considered atopic if the diameter of the skin reaction to at least one allergen exceeded 4 mm. The test was considered valid if the reaction to histamine was ≥ 6 mm according to the grading of skin prick test recommended by Atopy was determined using skin prick tests. The following battery of 24 aeroallergens common in Mexico City was used: http://pga.mbt.washington.edu) and genotyping data on 10 Mexicans and 38 Mexican-Americans with four grandparents born in Mexico for cosmopolitan haplotype tagging SNPs identified by the NIEHS Environmental Genome Project based on resequencing a representative sample of the U.S. population (http://egp.gs.washington.edu). We identified the common haplotypes using PHASE (http://snp500cancer.nci.nih.gov). TNF and LTA are small genes located consecutively in a 6 kb region on chromosome 6, and linkage disequilibrium in this region is high. We selected six tagging SNPs—TNF-1031 (rs1799964), TNF-857 (rs1799724), TNF-308 (rs1800629), TNF-238 (rs361525), LTA-379 (rs2239704), and LTA NcoI (rs909253)—to cover the whole region, including all the common SNPs in the regulatory and coding regions of the genes with known functional importance . We obtained genotypes for the ng Assay . Primersy system , provideng Assay . PrimersNonparentage was ascertained with a set of short-tandem repeats  analyzed using Pedcheck software  . A totalWe used a log-linear likelihood approach to analyze associations between asthma and individual SNPs . The logTNF and LTA polymorphisms influenced the degree of atopy, as assessed by the number of positive skin tests out of 24 performed, we used the polytomous logistic method of Kistner and Weinberg to estimate the linkage and association between TNF and LTA polymorphisms and atopy  to analyze associations between asthma and TNF and LTA haplotypes. HAPLIN is an extension of the log-linear model from a single locus to loci with multiple haplotypes with unknown phase  and cockroach (43.1%). Although only 5.8% of mothers reported smoking during pregnancy, 32.6% of cases lived with a smoking parent in early childhood (before 2 years of age) and 50.4% were currently exposed to parental smoking.Clinical characteristics of the asthmatic children with genotyping data are presented in TNF-308 and TNF-238 polymorphisms were relatively rare in our Mexican population . The frequency distributions for all mating types for TNF and LTA polymorphisms are presented in Supplemental http://www.ehponline.org/docs/2007/9740/suppl.pdf). Hardy-Weinberg equilibrium (p > 0.1) was confirmed for all six tagging SNPs in the parents. Pairwise linkage disequilibrium coefficients, D, and r2, between LTA and TNF SNPs, calculated using FBAT (LTA-379 and LTA NcoI polymorphisms (r2 = 0.50), and weak linkage disequilibrium between the LTA-379 and TNF-857 polymorphisms (r2 = 0.39), the TNF-1031 and TNF-238 polymorphisms (r2 = 0.25), the LTA NcoI and TNF-857 polymorphisms (r2 = 0.20), and the LTA-379 and TNF-1031 polymorphisms (r2 = 0.16). The r2 values for all other SNP pairs were < 0.1.The minor allele frequency and genotype frequency distributions of the six tagging SNPs are shown in ing FBAT  are showLTA was associated with asthma  = 1.54; 95% confidence interval (CI), 1.04–2.28; p = 0.031; false discovery rate = 0.12] relative to homozygotes for the major G allele .Neither of the two SNPs in h asthma . For TNFG allele . Only thh asthma . ResultsLTA and TNF SNP with asthma stratified by exposure to a smoking parent in the home. Among cases without smoking parents in the home, the TNF-308A allele and TNF-238A alleles showed increased risk of asthma  . Among cases with smoking parents in the home, none of the six LTA and TNF polymorphisms were associated with asthma  . The p-vh asthma .TNF-308A allele, exhibited an increased risk of asthma of borderline statistical significance . Among cases with non-smoking parents, carrying one copy of the ht6 (CACCGA) haplotype containing the TNF-238A allele or one copy of the ht5 haplotype exhibited an increased risk of asthma . The false discovery rate was 0.03 for the ht5 and ht6 findings. Among cases with smoking parents, LTA and TNF haplotypes were not associated with asthma .We examined the association between individual TNF-308 polymorphism was significantly associated with an increased risk of childhood asthma, especially among children with nonsmoking parents. The TNF-238A allele and the haplotypes containing the TNF-308A allele or the TNF-238A allele were associated with an increased childhood asthma risk predominantly in children with nonsmoking parents.In this case–parent triad study in a Mexico City population with high lifetime exposure to ozone, we found that the A allele of the functionally relevant in vitro, ex vivo, in vivo, and genetic studies  in our Mexican population, although they are only 70 bp apart from each other within the class III region of the MHC on chromosome 6p. There is no strong evidence showing that the TNF-238 polymorphism has a direct effect on gene expression, although studies suggest that this region contains a strong repressor site (−280 to −172)  . HoweverBecause the etiology of asthma involves numerous environmental triggers, heterogeneous exposure to environmental stimuli among different populations may cause conflicting results across studies. The influence of genotypes on phenotypes may be different and even opposite at different levels of exposure . TherefoTNF gene, was more strongly linked to asthma among subjects who did not live with a smoker during infancy compared with those who lived with smokers (It is not unexpected to find differing associations between genetic polymorphisms and asthma susceptibility based on parental smoking exposure. We previously reported that the protective effect for the NQO1 Ser allele in GSTM1-null children was limited to those with nonsmoking parents . A recen smokers .TNF-308 polymorphism modified the effect of home exposure to smokers on respiratory illness-related school absence among children mostly without asthma (TNF-308A allele was associated with an increased risk of wheezing, especially among children living in low ozone communities (TNF polymorphisms and haplotypes were associated with childhood asthma susceptibility predominantly among children who did not live with smoking parents.A recent study showed that the t asthma . Anothermunities . In the TNF is a candidate gene for ozone-induced airway inflammation and hyperresponsiveness (Because siveness , we straTNF polymorphisms with asthma predominantly for children without smoking parents is that the combination of exposure to secondhand smoke and ozone, which both increase TNF production, overwhelms the smaller impact of TNF polymorphisms on TNF expression. Indeed, expression levels of TNF are significantly increased in mice and humans exposed to tobacco smoke (TNF polymorphisms. Conversely, in the absence of secondhand smoke exposure, the influence of TNF functional polymorphisms on TNF expression may be greater, which could explain our finding of stronger associations at lower levels of exposure. Of note, for CD14, a pattern recognition receptor in the endotoxin-induced immune response, associations between the CD14-159 polymorphism and asthma and atopy differ at high versus low levels of endotoxin exposure (A possible explanation for our finding of an association of co smoke . TNF hasco smoke  and ozonco smoke . Tobaccoco smoke  and poteco smoke . In combexposure .TNF polymorphisms and haplotypes were associated with childhood asthma susceptibility, especially among children without smoking parents. These results suggest that the effects of genetic variation in TNF may be more apparent at lower levels of exposure to substances such as secondhand smoke, which strongly influence TNF expression.In summary, we found that


We have developed a simulation-based approach to the analysis of shared homozygous chromosomal segments and have applied it to data on allele sharing among alcoholics in a single Collaborative Study on the Genetics of Alcoholism pedigree. Our assessment of sharing involved the use of a single-nucleotide polymorphism (SNP) marker map provided by Affymetrix.All 11 affected individuals in the selected pedigree shared 2 copies of an allele at 4 adjacent SNPs in a region on chromosome 5. Via simulation, we determined that the probability that such sharing is caused by mere chance is less than 0.0000001. After correcting for undocumented inbreeding, this probability rose to 0.0016. The probability that the shared segment emanates from a single ancestor and is unrelated to the affection status is less than 0.0000001 in the corrected pedigree. Haplotype association analysis and a search for a protective locus using unaffected individuals yielded no significant results.Homozygosity mapping results on chromosome 5 provide suggestive evidence of the region's role as one that may harbor a genetic determinant of alcoholism. Furthermore, the probabilities of chance homozygous allele sharing for the original and for the inbreeding-corrected pedigree provide insight into the impact that inbreeding can have on such calculations. Homozygosity mapping has proven to be an effective approach in the identification of chromosomal regions that harbor autosomal recessive genes that influence disease susceptibility . The intIn order to make compelling claims about the probability that individuals actually share a segment of a chromosome homozygous by descent, two issues must be addressed. First, one must compute the probability that observed sharing of homozygous alleles at adjacent loci actually reflects sharing of a common ancestral segment and not just identity-by-state (IBS) allele sharing of a type that can occur purely by chance. Second, one must determine the expected amount of identity-by-descent (IBD) sharing unrelated to the disease. We have developed and applied a simulation-based approach to the analysis of shared homozygous chromosomal segments and have applied it to data on allele sharing among alcoholics in a single Collaborative Study on the Genetics of Alcoholism (COGA) pedigree.In order to combat possible allelic and locus heterogeneity, we decided to examine allele sharing among individuals within a single pedigree, COGA pedigree number 10022 , the expected size of an autozygous segment shared among the affecteds in pedigree 10022 is less than 18.6 Mb (authors' unpublished data). In the provided microsatellite scan, a segment this long would, on average, be marked by only 2 adjacent microsatellites. Actual autozygous regions would then be difficult to distinguish from regions represented by 2 markers shared IBS. For this reason, we decided to use the denser single-nucleotide polymorphism (SNP) map. Specifically, we used the cleaned dataset provided by Affymetrix.We developed a program that simulates chromosome inheritance in a given pedigree and outputs the frequency of shared homozygous loci, autozygous segments, and segments of different lengths shared by a given group of related individuals in that pedigree. Our simulation studies can discern autozygosity from chance homozygosity. We can also infer the conditional probability that a given segment is autozygous when it is observed as homozygous. These simulations are tailored to the precise location, marker density, and marker informativity associated with the observed data. Because recombination rate affects the number and size of homozygous segments (author's unpublished data), we designed the simulation method so that it utilizes empirical recombination rates to model recombination events. In this study, we used empirical recombination rates provided by Affymetrix. The following is a step-by-step description of the simulation procedure:1) Assign each founder unique chromosomal identification (ID) numbers2) Use detailed empirical information about chromosome lengths3) At each locus of relevance, assign each founder marker locus alleles that supervene on the chromosome ID numbers using allele frequency information or strict assignment of particular alleles4) Simulate recombination to assign chromosomes to each offspring using detailed empirical information about recombination rates5) Tally IBS/IBD marker allele sharing by examining assigned allele states and underlying chromosomal ID numbers6) Repeat many times to estimate probabilities of IBS sharing, IBD sharing, and IBD sharing conditional on IBS sharing probabilities based on relative frequencies For the region on chromosome 5, beginning with locus 49, there appeared to be evidence of significant sharing: all 11 affected individuals shared 2 copies of an allele at 4 adjacent SNPs. Furthermore, 1 of the 2 unaffected individuals did not exhibit this pattern, and was not homozygous in this region. Unfortunately, the genotype of the second unaffected individual was incomplete. Some of the members who are neither affected nor unaffected  shared the haplotype and some did not. Individuals 857, 438, and 1132 each carry one copy of the haplotype, which explains the observed homozygosity of their offspring.One important issue concerns the probability that the shared haplotype comes from a single ancestor. In the case of pedigree 10022, it is impossible that the affected members each share 2 copies of segment emanating from a single ancestor within the pedigree. Either the frequency of the haplotype in the population is high, and it entered the pedigree through several pedigree founders, or inbreeding is present in the generations preceding the founders' generations. We attempted to assess levels of inbreeding among pedigree members by counting the number of homozygous loci in the offspring of the founder pair (268 and 997), and comparing it to simulated counts from arbitrary inbred pedigree structures, specifically, the offspring of siblings, cousins, and second cousins. The average proportion of homozygous loci in 4 offspring in pedigree 10022 (69.7%) was less than in the offspring of sibs (70.7%), but greater than the proportion in offspring of cousins (67.8%). Since unaccounted inbreeding affects the haplotype sharing analysis, all subsequent calculations were also performed for pedigree 10022 with an additional generation consisting of the parents of the original founders , which is depicted in Figure The significance of the observed chromosomal sharing was assessed by simulating the inheritance of marker alleles and chromosome segments in the pedigree. The simulations were performed 10,000,000 times using the provided marker allele frequencies and inter-marker distances. By tracking the number of chromosomal segments in the inbreeding-corrected pedigree, we were able to determine the probability that the shared segment on chromosome 5 emanated from a single ancestor within the pedigree, and is unrelated to the affection status. This probability was less than 0.0000001 ). This result suggests that we encountered such an unlikely pattern of sharing due to ascertaining a pedigree with a high density of affected members or that the shared haplotype is common in the population, and entered the pedigree through different ancestors. The fact that the married-ins in the second generation are all carriers of the haplotype supports the latter explanation. Simulation studies were also used to address this question. In analysis of the pedigree without the correction for inbreeding, the probability of observing the sharing pattern found in affected members of pedigree 10022 was less than 0.0000001. However, when we accounted for possible inbreeding, the probability rose to 0.0016.Our aim in this study was to trace the number and size of shared chromosomal segments emanating from a common ancestor within the pedigree. Therefore, the simulation method assigned founder alleles assuming linkage equilibrium. The presence of strong linkage disequilibrium would modify the relative frequencies of sharing. However, since this pedigree was ascertained from an outbred population, we assumed that the effect of linkage disequilibrium on the relative frequencies would be negligible compared to the effect caused by inbreeding.Our results suggest that members of the investigated pedigree exhibit chromosomal sharing patterns compatible with a pedigree in which all founders are siblings. The patterns of sharing are also possible with different pedigree structures that would influence chromosome segment sharing probabilities. More sophisticated extraction of information on the pattern of inbreeding would make the calculations more reliable. A method for accurately estimating the inbreeding coefficient has been recently proposed by Leutenegger et al. . HoweverThe low probabilities of IBS chromosomal sharing, as well as IBD chromosomal sharing of the segment containing the proposed "disease" locus suggest that the haplotype is relatively common in the population from which the pedigree is sampled. The high frequency of the haplotype among the affected individuals may not be due to the fact that this region is associated with alcoholism. It might instead harbor a locus influencing a different trait common to the family under study. Analysis of unaffected individuals could provide stronger evidence for the role of this chromosomal region in the genetic predisposition of alcoholism. Unfortunately, this pedigree does not contain many unaffected individuals whose haplotypes could be analyzed. The additional haplotype association analysis and the search for a protective allele yielded no significant results.The results of our analyses suggest that the genetic basis of alcoholism, even when considered in context of a single pedigree, is still very complex. Our analysis of shared homozygous segments in the original and in the inbreeding-corrected COGA pedigree suggests that there is a region of shared homozygosity that isn't likely to occur by chance. This segment may harbor an alcoholism susceptibility locus. Further, the observed sharing has low probability even when the pedigree is corrected for inbreeding.COGA: Collaborative Study on the Genetics of AlcoholismIBD: Identity by descentIBS: Identity by stateID: IdentificationSNP: Single-nucleotide polymorphismOL implemented the simulations method, carried out the analyses, and drafted the manuscript. NJS invented the simulation method and revised the manuscript. Both authors read and approved the final manuscript.


SLC11A1 is known to link infections, autoimmunity and cancers. A review is presented of the mechanisms by which a balance is maintained between infections caused by pathogens  and disorders resulting from (acute or chronic) inflammation, and of the interactions that determine how the initial innate immune system directs subsequent acquired immune responses in human populations Slc11a1 in mice encodes a polytopic integral 10–12 transmembrane protein, which is expressed exclusively in macrophages and polymorphonuclear leukocytes and neurons (This review discusses the role of the solute carrier family 11 member 1 protein (SLC11A1), formerly NRAMP1 , in linking infections, autoimmunity and cancer. Chronic inflammation is caused by a variety of factors including bacterial, viral and parasitic infections, chemical irritants and nondigestible particles. Chronic inflammation is associated with a predisposition to cancer and autoimmunity because the longer the inflammation persists, the higher the risk of associated autoimmunity and carcinogenesis. Immune cells involved in inflammation include leucocytes such as neutrophils, monocytes, macrophages and eosinophils. These cells provide the soluble factors that are thought to mediate the development of inflammation. When inflammation goes unchecked it often leads to chronic inflammation-associated diseases. 2+) levels  levels . Thus, sMycobacterim bovis infection are significantly more acidic than those formed in slc11a1-negative ones .SLC11A1 should lead to apoptosis, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. By contrast, transcriptional repression of SLC11A1 could lead to cell survival. SLC11A1 might influence immune responses to viral vector delivery systems 5 ac (gt)5 ac (gt)9 g  drives high gene expression, while the allele t(gt)5 ac (gt)5 ac (gt)10 g  drives low gene expression , at 0.1%, is similar to that in the African populations  and within intronic regions, thus suggesting that this gene might be differentially regulated in different species. One major difference at this locus between species includes the type and locations of transcription factor binding sites in the 5′UTR of SLC11A1. Of particular importance is the binding site for the activating transcription factor-3 (ATF-3; also known as an AP-1-like element, Homo sapiens, Pan troglodytes and Macaca mulatta, this sequence is absent in Mus musculus. In Rattus norvegicus and Pongo pygmaeus, the second cytosine is replaced by a thymine. ATF-3 is a negative regulator of TLR4 (SLC11A1 but it is unclear whether it is an active ATF-3 binding site in SLC11A1. The formation of Z-DNA structure is known to play an active role in modulating inhibitory chromatin structure (SLC11A1 it should repress transcriptional activation of SLC11A1; conversely, heterodimerization of ATF3 with other transacting factors should promote transcriptional activation of SLC11A1.Agents that activate macrophage together with host SLC11A1 alleles will exert different effects on SLC11A1 function, but how SLC11A1 functions help determine the outcome of infection by modulating the production of these cytokines in not known. Comparative analysis of the  of TLR4 , where iIt could be that transcriptional activation of SLC11A1 leads to apoptosis and cell death while transcriptional repression of SLC11A1 leads to cell proliferation and survival if unchecked could result in cancer and autoimmunity. The ATF-3 putative site in the promoter of SLC11A1 along with other transacting factors might have a regulatory role in modulating the effects of SLC11A1. This review has thus highlighted some of the mechanisms by which SLC11A1 might be mediating, influencing or modulating the outcome of these diseases and disorders in human populations.Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.


In order to more effectively promote fruit and vegetable intake among children and adolescents, insight into determinants of intake is necessary. We conducted a review of the literature for potential determinants of fruit and vegetable intake in children and adolescents.Papers were identified from Medline and PsycINFO by using all combinations of the search terms: "fruit(s) or vegetable(s)" and "children or adolescents". Quantitative research examining determinants of fruit and/or vegetable intake among children and adolescents aged 6–18 years were included. The selection and review process was conducted according to a four-step protocol resulting in information on country, population, design, methodology, theoretical basis, instrument used for measuring intake, statistical analysis, included independent variables, and effect sizes.Ninety-eight papers were included. A large number of potential determinants have been studied among children and adolescents. However, for many presumed determinants convincing evidence is lacking, mostly because of paucity of studies. The determinants best supported by evidence are: age, gender, socio-economic position, preferences, parental intake, and home availability/accessibility. Girls and younger children tend to have a higher or more frequent intake than boys and older children. Socio-economic position, preferences, parental intake, and home availability/accessibility are all consistently positively associated with intake.The determinants most consistently supported by evidence are gender, age, socio-economic position, preferences, parental intake and home availability/accessibility. There is a need for internationally comparative, longitudinal, theory-based and multi-level studies taking both personal and environmental factors into account.] for the relevant editorial.This paper is published as part of the special Pro Children series in the International Journal of Behavioral Nutrition and Physical Activity. Please see [ A large body of epidemiological evidence suggests that a high fruit and vegetable intake helps to promote health and to prevent chronic disease -4. In moInterventions to improve health-related behaviours should be tailored to the most important determinants or mediators of these behaviours ,14. To dThe present review is part of the Pro Children Project. The Pro Children Project is an international-study involving nine European countries aiming at assessing fruit and vegetable consumption among schoolchildren and their parents, as well as positively affecting determinants of children's consumption . Based oTo our knowledge, a comprehensive review of the literature specifically studying determinants of children's fruit and vegetable intake has not previously been undertaken. The aim of the present paper is to provide a comprehensive review of potential determinants of fruit and vegetable intake in children and adolescents.Papers were identified by searching Medline  and PsycINFO . The search was conducted using all combinations of the search terms: fruit(s) or vegetable(s) and children or adolescents. The search from the databases identified 5,279 papers. The papers were screened by thorough reading of titles, abstracts, or full papers using the criteria for inclusion and exclusion specified below  the analyses were explicitly testing the relevance of one or more specified theoretical models, 2) the rationale for the analytical approach was based on a specified theoretical framework, or 3) the analyses were conducted without theoretical considerations. Some papers included multiple dependent variables or analysed multiple age groups. For these papers, the summary scheme contained information only for those variables and/or age-groups relevant for the present review.To evaluate the internal and external validity of the included papers, specific information on design and methodology were extracted and documented in a separate validity assessment scheme.An overall evidence scheme was produced including information on associations and significance for all variables involved in any paper. For each variable, the evidence was assessed across all papers included in the review. The identified variables were grouped into socio-demographic factors, personal factors, family-related factors, friends-related factors, school-related factors, meal patterns, TV watching, and eating fast food.To create a full overview of the included literature a summary table was constructed containing information on country, population, design, theoretical basis, instrument applied for measuring intake, statistical analysis, independent variables, significant associations identified, and effect sizes.Ninety-eight papers matched all inclusion criteria and were included in the review. The first identified paper was from 1958. The review revealed a marked rise during the recent years in number of papers on determinants of children and adolescents' fruit and vegetable consumption.From the evaluation of the design and methodology of each paper table  the main• Forty-eight (49%) of the included papers are based on US study populations• Eight papers (7%) use data from longitudinal studies• Four papers (4%) are explicitly testing the relevance of a theory while 12 papers (12%) are based on a theoretical framework• In 24 papers (25%) the sample is smaller than 500 individuals• Within 81 papers (83%) the samples are not representative for the age group at large in the country of study, or insufficient information is available for assessing representativeness.• In 58 papers (59%) the instrument applied for measuring fruit and/or vegetable intake is a food frequency questionnaire; 15 papers (15%) use one or several 24-hour-recalls; 16 papers (16%) use food diaries/records. Nine papers (9%) use other instruments, e.g. 'eating fruits and vegetables on the previous day (yes/no)' and 'type of food eaten for snack'.• Six papers (6%) analyse only fruit intake and one paper (1%) is on vegetable intake solely. In 47 papers (48%) fruit and vegetable intake is analysed separately. Thirty papers (31%) analyse a combined measure of fruit and vegetable intake while nine papers (9%) include analyses on both separate and combined measurements. Consumption of single items of specific fruit and/or vegetables  is analysed in five papers (5%).• Twenty-six papers (27%) are characterised by providing no information on validity of applied measurements and 31 papers (32%) only provide reference(s) to former publications. In 41 papers (42%) validity is assessed for all or some items/scales applied.• In 54 papers (55%) multivariate analyses were conducted while 34 (35%) papers include univariate analyses. Nine papers (9%) only include descriptive analyses. For one paper (1%) the type of analysis is not specified.Table Gender differences in fruit and vegetable consumption were studied in 49 papers. Twenty-seven of these found that girls tend to have a higher or more frequent intake of fruit and/or vegetables than boys. Eighteen papers observed no differences between boys and girls and four papers observed the highest or most frequent intake among boys. No systematic differences in age groups or instrument used for measuring fruit and/or vegetable intake exist between papers that identified gender differences in fruit and vegetable consumption and those that did not. However, a systematic difference seems to exist according to geographical region. The majority of the papers that analysed the effect of gender were either from the US or Europe. Of the 18 US papers only six found a difference in intake among boys and girls while 14 of the 17 European papers identified a gender difference. Thus, gender differences in intake of fruit and vegetables seem to be a phenomenon more prevalent among children and adolescents in European countries compared to children and adolescents in the US.The relevance of age (or grade) was studied in 22 papers. Ten of these papers found that fruit and vegetable consumption decreases with increasing age. In nine papers no effect of age was observed. In general, these nine papers are not characterised by narrower age/grade ranges or more extensive confounder control than those papers that did identify an association between age and fruit and vegetable intake. However, the majority of the papers that identified a negative association for age measured fruit and vegetable intake by a food frequency questionnaire (nine out of ten papers) while 24-hour recalls/food records were the dominant instrument applied among the papers that did not identify an association for age (six out of nine papers). This finding suggests that all age groups may eat the same amounts of fruit and vegetables but that the younger age groups eat fruit and vegetables more frequently than the older age groups or that there is an age-related response bias by assessment instrument used. Additionally, all of the six European papers that analysed the effect of age observed a negative association. In contrast, most of the US papers that analysed the effect of age found no association with intake (seven out of ten papers). Thus, decreasing intake of fruit and vegetables with increasing age of children and adolescents seems to be more related to European societies than to the US. Three papers found a positive association between age and intake of fruit and vegetables. In these three papers slightly different analyses were conducted on the same Tasmanian data set.Forty-six of the identified papers examined the influence of socioeconomic position (SEP). Within these papers, a large number of operationalisations of SEP were applied. Still, the findings are consistent. Low SEP is associated with low or less frequent intake of fruit and vegetables, and especially for family income (positive association in seven out of 14 papers), parental occupation (positive association in nine out of 11 papers) and parental education (positive association in 11 out of 11 papers) the association is well documented. For father's occupation specifically, two papers found a positive association, two papers found no association, and one paper found that high SEP students ate fruit more frequently at home while the most frequent intake of fruit at school was seen among low SEP students. The influence of mother's occupation was studied by two papers. Both found no association with fruit and vegetable consumption. The specific influence of father's education was studied in six papers of which only one identified a positive association. The influence of mother's education was analysed in eight papers of which four found higher or more frequent intake of fruit and/or vegetables with increasing educational level of mother. One longitudinal study, conducted in China, reported a negative association for mothers' educational level. Other examples of operationalisations of SEP that have been included in papers on fruit and vegetable consumption among children and adolescents are family material affluence (positive association in three out of four papers), student educational status (positive association in three out of three papers), several combined measurements (negative association in one out of two papers), and contextual measurements based on neighbourhoods or school catchments areas (positive association in six out of ten papers). No general systematic differences in age of study populations exist between those papers that identify an association between SEP and intake of fruit and/or vegetables among children and adolescents and those papers that do not. Also no clear systematic differences seem to exist according to geographical region. This is partly due to the fact that a number of papers each include two or more separate measurements for SEP for which different associations with intake of fruit and vegetables are observed.The literature on the influence of race/ethnicity is highly dominated by studies conducted with a US study population. The significant associations found (13 out of 19 studies) are inconsistent, depending on which ethnic groups were compared and often different patterns of consumption were observed for fruit and vegetables.In addition, 11 of the papers on race/ethnicity differences do not adjust for SEP. Comparing results may therefore be difficult due to differences in adjustments for confounders. Two European papers on the influence of race/ethnicity have been identified. One British paper (adjusted for SEP) observed the most frequent intake of fruit and vegetables among Black adolescents compared to White and Asian adolescents. One Danish paper  found that immigrant children (not specified) consume fruit and vegetables more frequent than Danish children.The influence of urbanisation has been investigated in four papers. Three of these found that fruit and/or vegetable consumption is higher or more frequent among rural children and adolescents than among urban children and adolescents. One paper found no association.Preference is the personal factor that has been examined most extensively. Eleven papers analysed the influence of preferences, and in all 11 papers a positive association between preferences and children and adolescents' intake of fruit and/or vegetables was observed.For nutritional knowledge, six papers observed positive associations with fruit and/or vegetable intake, one paper observed a negative association, and one paper did not find any association. Positive associations were also observed for attitude (three out of three papers), intentions (two out of four papers), self-efficacy (six out of eight papers) and subjective norms (perception of others' attitude on own diet) (three out of three papers). Five papers analysed the influence of outcome expectations and in one paper a positive association was identified. Barriers for eating fruit and vegetables were studied in three papers by the use of combined measures, e.g. availability, self-efficacy, cost, taste, and appeal. One paper found the most frequent intake of fruit and vegetables among adolescents with low perceived barriers. Two papers found no association.Within the family arena, a positive association between parental intake of fruit and/or vegetables and children's fruit and/or vegetable consumption was observed in eight out of nine papers. Four of these papers analysed parent reported parental intake and six papers analysed child perceived parental intake . One of these papers included perceived parental intake in a combined measure of perceived intake of significant others . Within two of the papers the effect of parental intake was modified by home availability, i.e. there is a stronger positive association between parental intake and children's fruit and vegetable intake within families with a high availability of fruit and vegetables at home.Three out of three papers identified a positive association between child-reported home availability of fruit and vegetables (fruit and vegetables being present in the home) and children's intake of fruit and vegetables. Within one of these papers, the association was only observed among girls. The influence of parent-reported availability was also analysed by three papers. One paper found no association, one paper found a positive association for girls only, and one paper found a direct positive association for girls and an indirect effect for boys . One out of one paper observed a positive association for parent-reported home accessibility of fruit and vegetables (form and location of fruit and vegetables present in the home). Within the same paper, a positive association was identified for child-reported accessibility among girls. Two out of three papers observed a positive association for combined measures of parent-reported home availability and accessibility. Combined measures of child-reported home availability and accessibility was analysed in only one paper. Here, a positive association was identified.The influence of family structure on children's fruit and vegetable consumption was analysed in five papers. Four papers found that fruit and/or vegetable consumption is lower among children from single-parent families than among children from two-parent families. Within two of these papers the effect of family structure were not adjusted for SEP. One paper (adjusted for SEP) found no association.The influence of family size was studied in three papers which all found no association with children's fruit and vegetable consumption.Six papers studied the influence of shared family meals and within five of these a positive association with children's consumption of fruit and/or vegetables was observed. One paper found no association.The association between parenting style and children's intake of fruit and vegetables was studied in six papers. These papers included ten different operationalisations of parenting styles. Three papers identified an association with children's fruit and vegetable consumption. In these papers the highest or most frequent intake was seen for authoritative parenting style, maternal authoritative parenting style, paternal non-authoritative style, and parental monitoring.Three out of three papers found a positive association between fruit and/or vegetable consumption and parental support for eating fruit or vegetables/healthy eating.The influence of friends has been only sparsely investigated. Two out of three papers found that perceived friend intake is positively associated with fruit and/or vegetable consumption.A variety of different operationalisations of availability of fruit and vegetables and other foods at school have been analysed in seven papers. One US and one Norwegian paper found a positive association for fruit and vegetables being served as part of the school lunch/fruit being served as part of a school fruit programme. No association was found between school availability of fruit (sold in school stores) and students' fruit consumption in a study from Belgium-Flanders. The same study also found no association for vending machines (selection not specified) being present at school. Another paper found that the number of snack vending machines (not differentiated according to snack type) is negatively associated with students' intake of fruit and vegetables. A limited number of papers also showed that à la carte school lunch program availability is negatively associated with students' intake of fruit and vegetables (one out of one papers), and that fruit and vegetable intake is lower among students with access to snack-bar meals versus those students with access to school lunch meals (two out of two papers).Two US and one Finnish paper found that students who participate in school lunch programmes have a higher or more frequent intake of fruit and vegetables than students that do not participate.The influence of academic achievement was studied by three papers. In two of these, low academic achievement is associated with low frequency of fruit and vegetable consumption.The influence of school type was studied in four papers  of which two found that students from non-public schools have the highest or most frequent intake of fruit and/or vegetables.The literature on the influence of meal patterns includes different measurements. In one out of three papers meal frequency is positively associated with fruit and vegetable consumption . Significant associations for meal type were found in five out of five papers though the applied operationalisations varied , school breakfast vs. home breakfast, and weekday meals vs. weekend day meals).Four papers examined the effect of watching TV, and they all found that increasing hours of TV watching is associated with decreasing fruit and/or vegetable consumption.The influence of eating fast food was studied in four US papers. Of these, three papers found that eating fast food is associated with low or less frequent consumption of fruit and vegetables, while one paper found no associationTable A comprehensive summary table (table 5) containing detailed information on design, analysis, and results of included papers is electronically available .The research presented in this review shows that the scientific effort to identify determinants of fruit and vegetable consumption among children and adolescents has increased substantially over the past four decades growing from a total of 11 published papers from 1958 through 1986 to 54 papers published only since 2000. This increase follows the growing evidence supporting the health benefits from eating ample amounts of fruit and vegetables  fth percentile for the knowledge score reported significantly greater intake of fruit and vegetables compared to students in all other quantiles. As stated in the paper, this finding may be due to a ceiling effect as most students scored very high on the knowledge questions.For nutritional knowledge, one US study by Lytle et al. (2003)  found thIn conclusion, several of the few contradictory findings identified within the present review may be attributable to methodological bias, while others may reflect realities.No previous review comparable to the present review exists. Recently a systematic review was conducted by Blanchette & Brug (2005) . This reThis review has several strengths and limitations. Literature relevant for the present review was identified by searching Medline and PsycINFO. Inclusion of other databases may have led to identification of additional relevant papers that matched the inclusion criteria. For instance, it is rather surprising that no papers on industry promotions  as a determinant of fruit and vegetable intake in children and adolescents were identified by the search and selection criteria. However, bibliographies of relevant papers including reviews and methodological papers were searched thoroughly until no more relevant papers emerged. Due to the language criteria, relevant information published in other languages than English may have been missed. The review is strengthened by the systematic approach by which procedures for evaluation and categorisation of all included papers were standardised. Within this review only significant associations are considered. In the field of epidemiology, the criteria for evaluation of estimated associations is a topic of discussion, and one recommendation is not to base evaluations on statistical significance alone . PreferaAdolescence is a time period of rapid physical maturation and growth combined with a psychological and social development which is often accompanied by changes in social influences. As children move into adolescence family influences often decrease due to competing influences from other social settings . It coulThere has been a clear quality improvement of the literature on determinants of fruit and vegetable consumption among children and adolescents over the past 15 years. In recent years a number of papers with high internal and external validity have been published e.g. ,58,79,87,8779,87]A considerable part of the papers include analyses based on small study samples and many papers include samples that are non-representative or only representative of a restricted geographical area. Often the validity of the applied instruments are only considered very superficially or not mentioned at all. Insufficient confounder control also seems to be a problem in a number of papers. For example, in some papers the analyses are not adjusted for relevant socio-demographic factors such as gender, age and/or SEP. Other bias may be evident in the types of instruments used  by age of participant. Parts of the literature on determinants of fruit and vegetable consumption among children and adolescents are therefore characterised by several methodological problems that may affect internal and external validity of the generated results.Comparisons of results from the different papers may be problematic for several reasons. A large variety of approaches for conceptualising, operationalising, measuring, and coding the outcome variable(s) exists among the identified papers. Some papers consider frequency of intake, while others consider amount of intake, and while these may be inter-correlated and both lead to the desired enhancement of intake, they may have different determinants. For instance, increasing diversity of fruit in the child's home may increase the amount of intake, while it may have no influence on frequency of intake, if measured as daily versus less than daily. In contrast, it may also be hypothesised that an increase in frequency of intake may be accompanied by decreasing portions sizes. In terms of defining the outcome variables, the possibility for comparing results is also compromised by two other aspects: 1) In some papers fruit consumption and vegetable consumption is analysed as separate outcomes, whereas others conduct the analyses on one combined measure. Comparing results from papers applying different analytical approaches may therefore be difficult. Additionally, combined analyses of fruit and vegetable intake may hide the fact that eating fruit and vegetables respectively may be linked to different sets of determinants; 2) Comparing results from different papers may also be problematic due to the fact that some papers include potatoes in the vegetable measurement, while others do not. In almost half of the included papers that analyse vegetable consumption, insufficient information is provided to assess whether potatoes are included in the measurement of vegetable intake or not. It is therefore not possible to evaluate the bias introduced by different definitions of vegetables, when comparing results. Correspondingly, some papers include fruit juice in the fruit category, while others do not. In more than half of the included papers it is not possible to assess, whether fruit juice is included in the measurement of fruit consumption.The conceptual framework applied within the Pro Children project is one of the most comprehensive models applied within the research on fruit and vegetable consumption among children and adolescents. The present review shows that several areas in this model can be identified for which research is very limited or lacking:• Psychosocial behavioural theories have been applied most often. Still, relatively few personal factors have been analysed extensively.• Family-related factors have been investigated most extensively. However, this part of the literature is characterised by a large number of conceptually different factors that are each investigated only sparsely. Generally, and also in terms of establishing healthy food habits, parents function as important role models for their children. Parents are responsible for making healthy food items available and accessible to the child within the home and for supporting and encouraging the child to make healthy food choices overall. However, the present review shows that only for a very limited number of family-related factors, good evidence exists. To enable health promoters to make evidence based decisions, more studies on the influence of the family setting for influencing fruit and vegetable intake among children and adolescents are therefore needed.• An overview of the effectiveness of interventions conducted at schools to promote fruit and vegetable intake among school children reveals that multi-component approaches including active provision of fruit and vegetables at lunch are those approaches that have been most successful ,125. Sti• Papers on potential influences of national level factors are almost absent. There is an obvious lack of papers looking at international differences in predictors. Future international comparative surveys should enable investigations of national level factors of importance e.g. price levels, policy, guidelines, supply, and exposure to mass media and commercials.• Likewise, little research has been reported on the potential influence of community or neighborhood level factors. Future research should therefore study the influence of e.g. local access to fruit and vegetables through grocery stores, local food policies, exposure to mass media and commercials, and fruit and vegetable availability in leisure time facilities for children and adolescents, like for instance local sport clubs.Across the identified areas for which research is lacking, future research would benefit from improvements in design and methodology. Almost every paper identified within this review was based on cross sectional data, and the need for future longitudinal analyses of children and adolescents' fruit and vegetable consumption is evident.Half of the papers identified by this review are based on US study populations. There is therefore an obvious lack of knowledge concerning predictors of fruit and vegetable consumption among children and adolescents from other parts of the world.Only few papers apply a theoretical approach. Introducing more theory based research may lead to more systematic research designs that ensure sound analytical models with sufficient confounder control. In addition, the theoretical frameworks (if any) of the papers included in the present review are mostly psychosocial and do not consider more structural environmental influences like nutritional policies and availability of fruit and vegetable in the different settings that children and adolescents take part in.We strongly recommend that future studies keep a very broad and comprehensive theoretical scope, in order not to exclude important etiological components of importance for child and adolescent fruit and vegetable intake.Introducing new comprehensive theoretical models should be accompanied by multilevel analytical approaches from which contextual effects can be estimated . To dateDespite the lack of consistent evidence for many potential determinants of fruit and vegetable intake in children and adolescents, a few recommendations for practice can be provided based on the present review.First of all, since fruit and vegetable intake appears to decline with age among children and adolescents, the present review confirms that intervention efforts are indeed needed to promote fruit and vegetable intake across childhood and adolescence. Furthermore, interventions to promote fruit and vegetable intake should especially be aiming at reaching youth from lower SEP groups, and specific efforts should be made to also reach boys. Such interventions should aim at improving preferences for fruits and vegetables, for example by more frequent exposure  to fruitDespite presenting solid documentation of the influence of several factors on fruit and vegetable consumption among children and adolescents this review also reveals that a long list of factors have only been sparsely investigated, and for several areas research is totally absent. Although the quality improvement of the research on children and adolescents' fruit and vegetable intake has been pronounced during recent years, a number of methodology problems have been identified. There is a need for further internationally comparative studies. At best, these should be theory-based multi-level studies in which both personal and environmental factors  are considered within a longitudinal design although we do realise that exploring such a broad range of potential determinants comes with measurement problems (e.g. long questionnaires or single item assessment of various constructs) . Such fuThe author(s) declare that they have no competing interest.MR directed the overall study and wrote the manuscript by incorporating critical inputs from all authors. KIK, PD and JB conceived the study. MR, RK and EB conducted the literature search and MR, RK and PD participated in the selection and analysis of included papers. All authors contributed to the design of the analysis. All authors read and approved the final manuscript.The additional file includes the large summary table 5. This comprehensive summary table contains detailed information on design, analysis, and results of all papers included in the present review. It will be made available at Click here for file


Interleukin-2 (IL-2) treatment induces other cytokines such as tumour necrosis factor (TNF) TNF may mediate some of the anti-tumour activity of IL-2, but conversely, may contribute to its dose limiting toxicities. Cleaved extracellular domains of the p55 and the p75 TNF receptors (sTNF-R1 and R2) bind to and inhibit the biological activity of TNF in vitro, but may also act as carrier molecules. We have assayed TNF and sTNFR-1 and 2 in the plasma of advanced cancer patients, before and during treatment with IL-2. Plasma levels of TNF in 22 patients were not significantly different from 25 normal controls, but levels of sTNFR-1 and sTNFR-2 were higher (P < 0.001). Levels of TNF and both its soluble receptors were significantly increased in 13 patients receiving IL-2 therapy. Maximum induced levels of sTNFR-1 and sTNFR-2 correlated closely with maximum induced levels of TNF (P < 0.001), but peak levels of sTNFR-1 and two were achieved 24-48 h after peak TNF. Levels of TNF and sTNF-Rs did not correlate with toxicity. Treatment with IL-2 leads not only to induction of TNF but also soluble binding proteins at levels which may modulate its biological activity.