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SIRT1 and orthologous sirtuins regulate a universal mechanism of ageing and thus determine lifespan across taxa; however, the precise mechanism remains vexingly polemical. They also protect against many metabolic and ageing-related diseases by dynamically integrating several processes including autophagy, proteostasis, calorie restriction, circadian rhythmicity and metabolism. These sirtuins are therefore important drug targets particularly because they also transduce allosteric signals from sirtuin-activating compounds such as resveratrol into increased healthspan in evolutionarily diverse organisms. While many of these functions are apparently regulated by deacetylation, that mechanism may not be all-encompassing. Since gonadal signals have been shown to regulate ageing/lifespan in worms and flies, the present study hypothesized that these sirtuins may act as intermediary factors for steroid hormone signal transduction. Accordingly, SIRT1 and its orthologues, Sir2 and Sir-2.1, are shown to be veritable nuclear receptor coregulators that classically coactivate the oestrogen receptor in the absence of ligand; coactivation was further increased by 17β-oestradiol. Remarkably in response to the worm steroid hormone dafachronic acid, SIRT1 reciprocally coactivates DAF-12, the steroid receptor that regulates nematode lifespan. These results suggest that steroid hormones may co-opt and modulate a phyletically conserved mechanism of sirtuin signalling through steroid receptors. Hence, it is interesting to speculate that certain sirtuin functions including prolongevity and metabolic regulation may be mechanistically linked to this endocrine signalling pathway; this may also have implications for understanding the determinative role of gonadal steroids such as oestradiol in human ageing. At its simplest, this report shows evidence for a hitherto unknown deacetylation-independent mechanism of sirtuin signalling.

SIRT1 protects against several complex metabolic and ageing-related diseases (MARDs), and is therefore considered a polypill target to improve healthy ageing. Although dietary sirtuin-activating compounds (dSTACs) including resveratrol are promising drug candidates, their clinical application has been frustrated by an imprecise understanding of how their signals are transduced into increased healthspan. Recent work indicates that SIRT1 and orthologous sirtuins coactivate the oestrogen receptor/ER and the worm steroid receptor DAF-12. Here they are further shown to ligand-independently transduce dSTACs signals through these receptors. While some dSTACs elicit ER subtype-selectivity in the presence of hormone, most synergize with 17β-oestradiol and dafachronic acid respectively to increase ER and DAF-12 coactivation by the sirtuins. These data suggest that dSTACs functionally mimic gonadal steroid hormones, enabling sirtuins to transduce the cognate signals through a conserved endocrine pathway. Interestingly, resveratrol non-monotonically modulates sirtuin signalling, suggesting that it may induce hormesis, i.e. “less is more”. Together, the findings suggest that dSTACs may be informational molecules that use exploitative mimicry to modulate sirtuin signalling through steroid receptors. Hence dSTACs’ intrinsic oestrogenicity may underlie their proven ability to impart the health benefits of oestradiol, and also provides a mechanistic insight into how they extend healthspan or protect against MARDs.

The liver expresses batteries of cytoprotective genes that confer cellular resistance to oxidative stress and xenobiotic toxins, and protection against cancer and other stress-related diseases. These genes are mainly regulated by Nrf2, making this transcription factor a target for small molecule discovery to treat such diseases. In this report, we identified dietary polyphenolic antioxidants that not only activated these genes but also relieved Nrf2 repression by Keap1, a Cul3-dependent ubiquitin ligase adaptor protein that mediates its degradation. Analysis of postprandial liver RNA revealed a marked activation of both genes by all test polyphenols compared with controls. Nrf2 inhibition by RNA interference reduced polyphenol effects on its target gene expression. Our data suggest that polyphenols may induce cellular defense genes by derepressing Nrf2 inhibition by Keap1. We posit that this ability to derepress Nrf2 and reactivate its target genes may underlie the protection conferred by polyphenols against oxidative stress-related diseases.

Background Iron metabolism during pregnancy maintains fetal iron levels at the expense of the mother. The mechanism behind this regulation is still not clear despite recent advances. Here we examine the role of maternal and fetal Hfe, its downstream signaling molecule, hepcidin and dietary iron in the regulation of placental iron transfer.Design and Methods Hfe wild-type, knockout and heterozygote dams were fed iron deficient (12.5 ppm), adequate (50 ppm) and replete (150 ppm) iron diets and mated with heterozygote males to produce pups of all genotypes. Dams and pups were sacrificed at Day 18 of gestation; serum, placenta, body and liver iron parameters were measured. Protein and mRNA levels of various iron transporter genes were determined in duodenum, liver and placenta by Western blotting and real time PCR.Results Maternal liver iron levels were dependent on both dietary iron intake and Hfe genotype. Increasing iron levels in the maternal diet resulted in increased total iron in the fetus, primarily in the liver. However, fetuses of Hfe-knockout mothers showed further elevation of liver iron levels, concomitant with elevated expression of Tfr1, Dmt1 and Fpn in the placenta. Hfe-knockout fetuses that express low levels of liver hepcidin accumulated more iron in their liver than wild-type fetuses due to increased ferroportin levels in the placenta.Conclusions Maternal and fetal status, as well as dietary iron, is important in regulating iron transfer across placenta. Maternal Hfe regulates iron transfer by altering gene expression in the placenta. Fetal Hfe is important in regulating placental iron transfer by modulating fetal liver hepcidin expression.

Background Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood. Methods We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay. Results We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme. Conclusion Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases. General significance The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.

Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. Therefore biomarkers contain relational or contextual information about disease pathophysiology. Two broad pathways can be taken to identify biomarkers: a 'top-down', holistic approach that makes no assumptions about biomarker type, or the 'bottom-up' approach, which is hypothesis driven and relies on a priori information. Both approaches involve parallel or sequential methods that include genomic and proteomic profiling. Biomarker discovery and translational medicine owe much to isotopic techniques because these provide near-real-time information about disease status as diagnostics, in drug delivery and for monitoring treatment. Here, we provide an overview of recent developments and some insight into the future role of isotopes in biomarker discovery and disease therapy.

RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.

The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasion by the merozoite forms of the parasite. Py235 is encoded by a large, highly polymorphic gene family, members of which appear to be differentially transcribed. However, it is not clear how many variants are expressed at the protein level during an infection cycle and whether or not these variants are expressed selectively or combinatorially. Certain monoclonal antibodies to Py235 have been shown to attenuate parasite virulence upon passive transfer into mice, suggesting that this antigen or its derivatives may be useful vaccine candidates. To provide a basis for this, we sought to identify those variants that are recognised by the host immune system, and to establish the pattern of expression of the antigen in mice during infection. Using Py235 monoclonal antibodies as probes, we isolated distinct antigenic variants from an expression library, suggesting that the antigen repertoire is potentially large and that different Py235 variants may be produced during infection. The implications of these observations are discussed with respect to the ability of a cloned parasite line to express distinct antigenic variants in vivo.

Hepcidin is a liver-derived antimicrobial peptide that regulates iron absorption and is also an integral part of the acute phase response. In a previous report, we found evidence that this peptide could also be induced by toxic heavy metals and xenobiotics, thus broadening its teleological role as a defensin. However it remained unclear how its sensing of disparate biotic and abiotic stressors might be integrated at the transcriptional level. We hypothesized that its function in cytoprotection may be regulated by NFE2-related factor 2 (Nrf2), the master transcriptional controller of cellular stress defenses. In this report, we show that hepcidin regulation is inextricably linked to the acute stress response through Nrf2 signaling. Nrf2 regulates hepcidin expression from a prototypical antioxidant response element in its promoter, and by synergizing with other basic leucine-zipper transcription factors. We also show that polyphenolic small molecules or phytoestrogens commonly found in fruits and vegetables including the red wine constituent resveratrol can induce hepcidin expression in vitro and post-prandially, with concomitant reductions in circulating iron levels and transferrin saturation by one such polyphenol quercetin. Furthermore, these molecules derepress hepcidin promoter activity when its transcription by Nrf2 is repressed by Keap1. Taken together, the data show that hepcidin is a prototypical antioxidant response or cytoprotective gene within the Nrf2 transcriptional circuitry. The ability of phytoestrogens to modulate hepcidin expression in vivo suggests a novel mechanism by which diet may impact iron homeostasis., Graphical abstract fx1, Highlights • Nrf2 regulates HAMP transcription. • Phytoestrogens differentially induce HAMP transcription by Nrf2. • Post-prandial hepcidin induction reduces serum iron and transferrin saturation. • Nrf2 knockout results in iron-overload in mouse embryonic fibroblasts.

We investigated the potential of a new family of lipidic peptide dendrimers in protein transduction into cultured cells. Dendrimer-protein interaction was determined by gel retardation assays using purified recombinant protein. To assess intracellular protein delivery, two marker proteins were used: recombinant firefly luciferase and a Cy3-labeled monoclonal antibody to the c-myc proto-oncogene. Protein delivery was determined by luciferase assays and fluorescence microscopy, respectively. While there was minimal delivery of luciferase or antibody in the absence of the dendrimers, the latter increased protein delivery substantially. Luciferase delivery was concentration and cell type-dependent; the efficiency of delivery also varied with the number of terminal amino groups on the dendrimers. In previous reports, we showed that these dendrimers could be used for gene and drug delivery; the data we report herein suggest that they may also be capable of intracellular protein delivery. This finding has important implications for the use of these dendrimers in protein therapeutics and vaccinology.

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function.

TNFalpha has dramatic effects on iron metabolism contributing to the generation of hypoferraemia in the anaemia of chronic disease. Interestingly, TNFalpha is also synthesised and released within the intestinal mucosa, suggesting that this pro-inflammatory cytokine may play a role in regulating dietary iron absorption. To investigate this possibility, we stimulated intestinal Caco-2 cells with TNFalpha (10 ng/ml). In TNFalpha-treated cells, apical iron uptake was significantly decreased and this was accompanied by a reduction in divalent metal transporter protein and mRNA expression. Our data suggest that TNFalpha could regulate dietary iron absorption and that the apical transport machinery is the target for these actions.

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.

Glucose transport in Trypanosoma brucei is facilitated by a transporter that is kinetically markedly different from its mammalian homologue. In this regard, the trypanosomal transporter may be selectively targeted. We investigated the potential of a series of triazinyl derivatives as inhibitors of glucose transport in T. brucei. A graded response of glucose transporter inhibition by these compounds was observed, with Cibacron blue 3GA, CiB, being the most potent. This inhibited transport by up to 90% in a concentration-dependent manner, with an IC50 of about 19.4 microM. A Dixon plot of different concentrations of this triazine and the rate of transport suggested that inhibition may be simple and competitive. The inhibition constant Ki was 14.8 microM. Although cytochalasin B has been widely reported to inhibit glucose uptake by mammalian and other eukaryotic glucose transporters, it had no effect at all on the trypanosome transporter at concentrations equivalent to those of the triazines. This may suggest structural differences between the trypanosome and mammalian glucose transporters and also suggests that the triazine moiety may serve as a template for the design potent trypanocides targeted at the glucose transporter.

In the rodent malaria species, Plasmodium yoelii, a multi-gene family (Py235) encodes a 235 kDa rhoptry protein. This protein is believed to be involved in merozoite attachment and invasion of red blood cells. Only two members of Py235 have been sequenced so far. Using genomic DNA from the virulent P. yoelii YM line we have PCR amplified additional members of this gene family. These >8 kb full length clones have been cloned and sequenced. Based on differences within the tri-amino acid repeat structure at the C-terminal end of the Py235 protein, it has been possible to divide the multi-gene family into subtypes. The protein translations of five full-length genes (representing four different subtypes) were compared. While there was a high level of amino acid identity at the C-terminal end of these proteins, the N-terminal region revealed a great deal of sequence diversity. Critically, certain residues appeared to be conserved notably seven out of eight cysteines. Comparison of two full-length genes of a particular sub-type shows >99% amino acid identity at the protein level, implying that very closely related genes exist within the parasite genome. We have used this new sequence information to compare the distribution of Py235 in the virulent YM and avirulent 17X lines of P. yoelii. Our results indicate that while the overall distribution of Py235 genes is broadly conserved between the two lines, significant differences exist when individual subtypes are compared.

We carried out a genome-wide association study of hemoglobin levels in 16,001 individuals of European and Indian Asian ancestry. The most closely associated SNP (rs855791) results in nonsynonymous (V736A) change in the serine protease domain of TMPRSS6 and a blood hemoglobin concentration 0.13 (95% CI 0.09-0.17) g/dl lower per copy of allele A (P = 1.6 x 10(-13)). Our findings suggest that TMPRSS6, a regulator of hepcidin synthesis and iron handling, is crucial in hemoglobin level maintenance.

Background Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation. Methods To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression. Results Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional. Conclusions From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.

Genetic variation underlies phenotypic diversity and complex quantitative traits including heritable diseases. We hypothesized that such variation may underlie or determine intrinsic inter-individual differences in iron metabolism and may also play a role in variable phenotypes associated with iron-related diseases. Using hepcidin as a marker of iron homeostasis, we assessed sequence variation and the transcription potencies of promoter haplotypes for both hepcidin genes mhepc1 and mhepc2 from different strains of inbred mice. We found several single nucleotide polymorphisms (SNPs) within the promoters of both genes on one hand, and between strains on the other. With luciferase as reporter, we also found significant variation in the basal transcription of both genes. A regulatory SNP constituting an E-box in the promoter of mhepc1 caused further expression level variation and transactivation by Upstream Stimulatory Factor, USF. Inter-strain variation in hepcidin expression correlated with established phenotypic differences in iron loading in these mice. As hepcidin is critically required for iron metabolism, we posit that variation in its expression may be a quantitative trait which determines differences in iron handling within and between mouse strains, and that this may also apply to humans. Thus, regulatory variation in hepcidin expression may be just as important as structural variation or mutations within its coding sequence.

The Ity/Lsh/Bcg locus encodes the macrophage protein Slc11a1/Nramp1, which protects inbred mice against infection by diverse intracellular pathogens including Leishmania, Mycobacterium, and Salmonella. Human susceptibility to infectious and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and tuberculosis, shows allelic association with a highly polymorphic regulatory, Z-DNA–forming microsatellite of (GT/AC)n dinucleotides within the proximal SLC11A1 promoter. We surmised that cis-acting allelic polymorphisms may underlie heritable differences in SLC11A1 expression and phenotypic variation in disease risk. However, it is unclear what may underlie such variation in SLC11A1 allele expression. Here we show that hypoxia-inducible Factor 1 (HIF-1) regulates allelic variation in SLC11A1 expression by binding directly to the microsatellite during macrophage activation by infection or inflammation. Targeted Hif-1α ablation in murine macrophages attenuated Slc11a11 expression and responsiveness to S typhimurium infection. Our data also showed that HIF-1 may be functionally linked to complex prototypical inflammatory diseases associated with certain SLC11A1 alleles. As these alleles are highly polymorphic, our finding suggests that HIF-1 may influence heritable variation in SLC11A1-dependent innate resistance to infection and inflammation within and between populations. This report also suggests that microsatellites may play critical roles in the directional evolution of complex heritable traits by regulating gene expression phenotypes.

The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5- and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.

Hepcidin is the presumed negative regulator of systemic iron levels; its expression is induced in iron overload, infection, and inflammation, and by cytokines, but is suppressed in hypoxia and anemia. Although the gene is exquisitely sensitive to changes in iron status in vivo, its mRNA is devoid of prototypical iron-response elements, and it is therefore not obvious how it may be regulated by iron flux. The multiplicity of effectors of its expression also suggests that the transcriptional circuitry controlling the gene may be very complex indeed. In delineating enhancer elements within both the human and mouse hepcidin gene promoters, we show here that members of the basic helix-loop-helix leucine zipper (bHLH-ZIP) family of transcriptional regulators control hepcidin expression. The upstream stimulatory factor 2 (USF2), previously linked to hepcidin through gene ablation in inbred mice, appears to exert a polar or cis-acting effect, while USF1 may act in trans to control hepcidin expression. In mice, we found variation in expression of both hepcidin genes, driven by these transcription factors. In addition, c-Myc and Max synergize to control the expression of this hormone, supporting previous findings for the role of this couple in regulating iron metabolism. Transcriptional activation by both USF1/USF2 and c-Myc/Max heterodimers occurs through E-boxes within the promoter. Site-directed mutagenesis of these elements rendered the promoter unresponsive to USF1/USF2 or c-Myc/Max. Dominant-negative mutants of USF1 and USF2 reciprocally attenuated promoter transactivation by both wild-type USF1 and USF2. Promoter occupancy by the transcription factors was confirmed by DNA-binding and chromatin immunoprecipitation assays. Taken together, it would appear that synergy between these members of the bHLH-ZIP family of transcriptional regulators may subserve an important role in iron metabolism as well as other pathways in which hepcidin may be involved.

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.

Hepcidin is a small acute phase peptide that regulates iron absorption. It is induced by inflammation and infection, but is repressed by anaemia and hypoxia. Here we further reveal that hepcidin transcription also involves interactions between functional metal response elements (MREs) in its promoter, and the MRE-binding transcription factor-1. Analysis of hepcidin mRNA and protein levels in hepatoma cells suggests that its expression may be regulated by divalent metal ions, with zinc inducing maximal effects on hepcidin levels. These data suggest that this peptide may be a pleiotropic sensor of divalent metals, some of which are xenobiotic environmental toxins.

The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes. Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays. The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport. The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane. This observation is consistent with a facilitated transporter. There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes. In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose. Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport. The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion. A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.