Repurposing ethyl bromopyruvate as a broad-spectrum antibacterial.

BACKGROUND: The emergence of drug-resistant bacteria is a major hurdle for effective treatment of infections caused by Mycobacterium tuberculosis and ESKAPE pathogens. In comparison with conventional drug discovery, drug repurposing offers an effective yet rapid approach to identifying novel antibiotics. METHODS: Ethyl bromopyruvate was evaluated for its ability to inhibit M. tuberculosis and ESKAPE pathogens using growth inhibition assays. The selectivity index of ethyl bromopyruvate was determined, followed by time-kill kinetics against M. tuberculosis and Staphylococcus aureus. We first tested its ability to synergize with approved drugs and then tested its ability to decimate bacterial biofilm. Intracellular killing of M. tuberculosis was determined and in vivo potential was determined in a neutropenic murine model of S. aureus infection. RESULTS: We identified ethyl bromopyruvate as an equipotent broad-spectrum antibacterial agent targeting drug-susceptible and -resistant M. tuberculosis and ESKAPE pathogens. Ethyl bromopyruvate exhibited concentration-dependent bactericidal activity. In M. tuberculosis, ethyl bromopyruvate inhibited GAPDH with a concomitant reduction in ATP levels and transferrin-mediated iron uptake. Apart from GAPDH, this compound inhibited pyruvate kinase, isocitrate lyase and malate synthase to varying extents. Ethyl bromopyruvate did not negatively interact with any drug and significantly reduced biofilm at a 64-fold lower concentration than vancomycin. When tested in an S. aureus neutropenic thigh infection model, ethyl bromopyruvate exhibited efficacy equal to that of vancomycin in reducing bacterial counts in thigh, and at 1/25th of the dosage. CONCLUSIONS: Ethyl bromopyruvate exhibits all the characteristics required to be positioned as a potential broad-spectrum antibacterial agent.

Lectin-Based Assay for Glycoform-Specific Detection of α2,6-sialylated Transferrin and Carcinoembryonic Antigen in Tissue and Body Fluid.

Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed, Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.

Enhancing Glioblastoma-Specific Penetration by Functionalization of Nanoparticles with an Iron-Mimic Peptide Targeting Transferrin/Transferrin Receptor Complex.

Treatment of glioblastoma (GBM) remains to be the most formidable challenge because of the hindrance of the blood-brain barrier (BBB) along with the poor drug penetration into the glioma parenchyma. Nanoparticulate drug delivery systems (DDS) utilizing transferrin (Tf) as the targeting ligand to target the glioma-associated transferrin receptor (TfR) had met the problem of loss of specificity in biological environment due to the high level of endogenous Tf. Here we conjugated CRT peptide, an iron-mimicry moiety targeting the whole complex of Tf/TfR, to poly(ethylene glycol)-poly(l-lactic-co-glycolic acid) nanoparticles (CRT-NP), to open a new route to overcome such obstacle. High cellular associations, advanced transport ability through the BBB model, and penetration in 3-dimensional C6 glioma spheroids in vitro had preliminarily proved the advantages of CRT-NP over Tf-nanoparticle conjugates (Tf-NP). Compared with Tf-NP, NP, and Taxol, paclitaxel-loaded CRT-NP (CRT-NP-PTX) displayed a superior antiproliferation effect on C6 glioma cells and stronger inhibitory effect on glioma spheroids. Favored pharmacokinetics behavior and enhanced accumulation in glioma foci was observed, together with a much deeper distribution pattern in glioma parenchyma compared with unmodified nanoparticles and Tf-NP. Eventually, mice treated with CRT-NP-PTX showed a remarkably prolonged median survival compared to those treated with Taxol, NP, or Tf-NP. In conclusion, the modification of CRT to nanoparticles holds great promise for enhancement of antiglioma therapy.

The glycation site specificity of human serum transferrin is a determinant for transferrin's functional impairment under elevated glycaemic conditions.

The mechanisms involving iron toxicity in diabetes mellitus are not completely understood. However, the spontaneous reaction of reducing sugars with protein amino groups, known as glycation, has been shown to compromise the action of Tf (transferrin), the systemic iron transporter. In order to understand the structural alterations that impair its function, Tf was glycated in vitro and the modification sites were determined by MS. Iron binding to glycated Tf was assessed and a computational approach was conducted to study how glycation influences the iron-binding capacity of this protein. Glycated Tf samples were found to bind iron less avidly than non-modified Tf and MS results revealed 12 glycation sites, allowing the establishment of Lys534 and Lys206 as the most vulnerable residues to this modification. Their increased susceptibility to glycation was found to relate to their low side-chain pKa values. Lys534 and Lys206 participate in hydrogen bonding crucial for iron stabilization in the C- and N-lobes of the protein respectively, and their modification is bound to influence iron binding. Furthermore, the orientation of the glucose residues at these sites blocks the entrance to the iron-binding pocket. Molecular dynamics simulations also suggested that additional loss of iron binding capacity may result from the stereochemical effects induced by the glycation of lysine residues that prevent the conformational changes (from open to closed Tf forms) required for metal binding. Altogether, the results indicate that Tf is particularly vulnerable to glycation and that this modification targets spots that are particularly relevant to its function.

Regulation of EGF-stimulated EGF receptor endocytosis during M phase.

It has been generally accepted that endocytosis is inhibited during mitotic phase (M phase) as a means to insulate the cell from outside influences. Many endocytic/trafficking proteins are present during M phase, but are associated with partners that are distinct from those involved in trafficking pathways. These findings have led to the 'moonlighting' hypothesis. However, all these findings are based on the study of fluid-phase and constitutive endocytosis. Here, we used epidermal growth factor receptor (EGFR) as a model system to study ligand-induced receptor endocytosis in M phase. We found that EGF-induced EGFR endocytosis still occurs during M phase, but follows different kinetics. EGF-induced EGFR endocytosis is delayed/inhibited for a few minutes and is slower in M phase, especially at metaphase. However, consistent with previous reports, transferrin endocytosis is inhibited under the same conditions. We further showed that EGFR endocytosis is differentially regulated during the cell cycle: dependent on EGFR kinase activation in M phase, but independent of EGFR kinase activation in interphase. We conclude that cells have adopted a system for selective endocytosis in M phase.

Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

BACKGROUND INFORMATION: Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. RESULTS: Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCßII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCßII but not PKCßI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCßII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. CONCLUSIONS: This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking.

Use of spectroscopic, zeta potential and molecular dynamic techniques to study the interaction between human holo-transferrin and two antagonist drugs: comparison of binary and ternary systems.

For the first time, the binding of ropinirole hydrochloride (ROP) and aspirin (ASA) to human holo-transferrin (hTf) has been investigated by spectroscopic approaches (fluorescence quenching, synchronous fluorescence, time-resolved fluorescence, three-dimensional fluorescence, UV-vis absorption, circular dichroism, resonance light scattering), as well as zeta potential and molecular modeling techniques, under simulated physiological conditions. Fluorescence analysis was used to estimate the effect of the ROP and ASA drugs on the fluorescence of hTf as well as to define the binding and quenching properties of binary and ternary complexes. The synchronized fluorescence and three-dimensional fluorescence spectra demonstrated some micro-environmental and conformational changes around the Trp and Tyr residues with a faint red shift. Thermodynamic analysis displayed the van der Waals forces and hydrogen bonds interactions are the major acting forces in stabilizing the complexes. Steady-state and time-resolved fluorescence data revealed that the fluorescence quenching of complexes are static mechanism. The effect of the drugs aggregating on the hTf resulted in an enhancement of the resonance light scattering (RLS) intensity. The average binding distance between were computed according to the forster non-radiation energy transfer theory. The circular dichroism (CD) spectral examinations indicated that the binding of the drugs induced a conformational change of hTf. Measurements of the zeta potential indicated that the combination of electrostatic and hydrophobic interactions between ROP, ASA and hTf formed micelle-like clusters. The molecular modeling confirmed the experimental results. This study is expected to provide important insight into the interaction of hTf with ROP and ASA to use in various toxicological and therapeutic processes.

Transferrin receptor mediates uptake and presentation of hepatitis B envelope antigen by T lymphocytes.

Human activated T lymphocytes expressing class II molecules are able to present only complex antigens that bind to their own surface receptors, and thus can be captured, internalized, and processed through the class II major histocompatibility complex processing pathway. We have used the antigen-presenting T cell system to identify the viral receptor used by hepatitis B virus (HBV) to enter cells, as well as the sequence of HB envelope antigen (HBenvAg) involved in this interaction. Results show that both CD4+ and CD8+ T clones can process and present HBenvAg to class II-restricted cytotoxic T lymphocytes and that the CD71 transferrin receptor (TfR) is involved in efficient HBenvAg uptake by T cells. Moreover, we provide evidence that the HBenvAg sequence interacting with the T cell surface is contained within the pre-S2 region. Since TfR is also expressed on hepatocytes, it might represent a portal of cellular entry for HBV infection. This system of antigen presentation by T cells may serve as a model to study both lymphocyte receptors used by lymphocytotropic viruses and viral proteins critical to bind them.

Insect transferrin functions as an antioxidant protein in a beetle larva.

In insects transferrin is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone regulated protein. Here, a novel functional role for insect transferrin as an antioxidant protein is demonstrated. Stressors, such as heat shock, fungal challenge, and H(2)O(2) exposure, cause upregulation of the white-spotted flower chafer Protaetia brevitarsis (Coleoptera: Scarabaeidae) transferrin (PbTf) mRNA in the fat body and increases PbTf protein levels in the hemolymph. RNA interference (RNAi) treated PbTf reduction causes increased iron and H(2)O(2) levels in the hemolymph and results in induction of apoptotic cell death in the fat body during exposure to stress. The observed effects of PbTf RNAi suggest that PbTf inhibits stress-induced apoptosis by diminishing the Fenton reaction via the binding of iron, thus supporting an antioxidant role for PbTf in stress responses.

Inhibition of transferrin iron release increases in vitro drug carrier efficacy.

Transferrin (Tf) conjugates of CRM107 are currently being tested in clinical trials for treatment of malignant gliomas. However, the rapid cellular recycling of Tf limits its efficiency as a drug carrier. We have developed a mathematical model of the Tf/TfR trafficking cycle and have identified the Tf iron release rate as a previously unreported factor governing the degree of Tf cellular association. The release of iron from Tf is inhibited by replacing the synergistic carbonate anion with oxalate. Trafficking patterns for oxalate Tf and native Tf are compared by measuring their cellular association with HeLa cells. The amount of Tf associated with the cells is an average of 51% greater for oxalate Tf than for native Tf over a two hour period at Tf concentrations of 0.1 nM and 1 nM. Importantly, diphtheria toxin (DT) conjugates of oxalate Tf are more cytotoxic against HeLa cells than conjugates of native Tf. Conjugate IC(50) values were determined to be 0.06 nM for the oxalate Tf conjugate vs. 0.22 nM for the native Tf conjugate. Thus, we show that inhibition of Tf iron release improves the efficacy of Tf as a drug carrier through increased association with cells expressing TfR.

A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML.

Limited success of antisense oligonucleotides (ASO) in clinical anticancer therapy calls for more effective delivery carriers. The goal of this study was to develop a nanoparticle system for delivery of ASO G3139, which targets mRNA of antiapoptotic protein Bcl-2, to acute myeloid leukemia (AML) cells. The synthesized nanoparticle Tf-LPN-G3139 contained a small molecular weight polyethylenimine and two cationic lipids as condensing agents, with transferrin on its surface for selective binding and enhanced cellular uptake. The optimized nitrogen to phosphate (N/P) ratio was 4 to achieve small particle size and high G3139 entrapment efficiency. The Tf-LPN-G3139 exhibited excellent colloidal stability during storage for at least 12 weeks and remained intact for 4 hours in nuclease-containing serum. The cellular uptake results showed extensive internalization of fluorescence-labelled G3139 in MV4-11 cells through Tf-LPN. Following transfection, Tf-LPN-G3139 at 1 µM ASO level induced 54% Bcl-2 downregulation and >20-fold apoptosis compared to no treatment. When evaluated in mice bearing human xenograft AML tumors, Tf-LPN-G3139 suppressed tumor growth by ~60% at the end of treatment period, accompanied by remarkable pharmacological effect of Bcl-2 inhibition in tumor. In conclusion, Tf-LPN-G3139 is a promising nanoparticle system for ASO G3139 delivery to AML and warrants further investigations.

The acute-phase protein alpha 1-antitrypsin inhibits transferrin-receptor binding and proliferation of human skin fibroblasts.

Transferrin (Tf) is required for proliferation of most cells, because cellular iron uptake is mainly mediated by binding of Tf to its specific cell surface receptors (TfR). The acute-phase protein alpha 1-antitrypsin (alpha 1-AT) completely inhibits binding of diferric Tf to TfRs on human skin fibroblasts in a dose-dependent fashion. The inhibition is competitive as proved in equilibrium saturation binding and kinetic studies. In saturation binding experiments alpha 1-AT apparently increased the dissociation constant (KD), but did not change the maximal density of binding sites (Bmax). As shown in kinetic studies, this reduction of the affinity of Tf to its receptor caused by alpha 1-AT was due to a decrease of the association rate constant (k + 1), whereas the dissociation rate constant (k - 1) remained unchanged. Furthermore, alpha 1-AT almost completely prevented internalization of the Tf-TfR complex. These interactions demonstrated biological implication, as alpha 1-AT reduced the proliferation of human fibroblasts up to maximal 30% of control. The inhibitory potency of alpha 1-AT was already seen in physiologic concentrations; the maximal effect, however, was achieved at concentrations above the normal range, which are attained in the course of inflammation and infection. Therefore, we suppose that alpha 1-AT as an endogenous factor modulates the complex mechanism of fibrogenesis not only by its known antiproteolytic function but also by inhibiting the proliferation of fibroblasts.

Selection of tumor-specific internalizing human antibodies from phage libraries.

Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.

Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport.

Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.

Accelerated neuronal differentiation toward motor neuron lineage from human embryonic stem cell line (H9).

Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a cost-effective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are ∼1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

Characterization of a new monoclonal antibody (TR-19) against human transferrin receptor and its application in topographic study.

A new monoclonal antibody TR-19 (IgG2a) directed to the human transferrin receptor (CD71) was prepared after immunization of BALB/c mice with human non-T, non-B ALL cell line REH-6. This monoclonal antibody (mAb) reacted in immunofluorescence with all human cell lines tested. The reactivity with human peripheral blood leukocytes was observed only after stimulation with phytohemagglutinin (PHA). The protein precipitated by mAb TR-19 has an apparent molecular weight of 180 kDa under nonreducing and 90 kDa under reducing conditions. Sequential immunoprecipitation showed that mAb TR-19 recognizes the same antigen as the reference anti-transferrin receptor mAb OKT9. The mAb TR-19 coprecipitates the radioiodinated transferrin as a complex with its receptor. The mAb TR-19 with other three mAbs (OKT9, 5E9 and MEM-75) was used in the topographic study of the transferrin receptor. By competitive binding radioimmunoassay it was found that these mAbs recognize two distinct antigenic sites: One is specified by mAbs TR-19 and OKT9 and the second by mAbs 5E9 and MEM-75.

Effects of ethanol on the secretion of hepatic secretory protein in rat alcoholic liver injury.

It has been pointed out that one of the pathogenetic causes of alcoholic liver injury is the hepatocytic accumulation of exportable proteins due to a decrease in hepatic microtubules caused by acetaldehyde. To confirm and extend this secretory protein accumulation in the hepatocytes, the effects of alcohol treatment on the intracellular transport of secretory protein in the hepatocyte was studied using radioisotope-labeled leucine and fucose. Acute ethanol administration to rats did not show any effects on intrahepatocytic transport and secretion of transferrin. In alcohol pyrazole hepatitis rats, the secretion of transferrin labeled with both radioactive leucine and fucose into the serum was significantly delayed. Delaying in the secretion of fucose-labeled transferrin was more prominent than in leucine-labeled transferrin. This secretory inhibition was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled transferrin. At the time of the maximum inhibition of secretion, radioisotope labeled transferrin mainly retained in the Golgi apparatus. These results indicated that movement of secretory proteins along the secretory pathway impaired in alcoholic liver injury and that accumulation of the secretory proteins might play an important role in the development of alcoholic liver injury.

Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.

In Trypanosoma brucei, uptake of host transferrin is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor derived from the 2 expression site-associated genes 6 and 7 (ESAG6 and ESAG7). By using specific antibodies, it is shown here that T. evansi, a trypanosome species transmitted mechanically by biting flies, also expresses a transferrin receptor composed of ESAG6 and ESAG7. The cellular uptake of transferrin in T. evansi is completely inhibited with anti-T. brucei (ESAG6/7 heterodimer) antibodies. The demonstration of a functional ESAG6/7 transferrin receptor in T. evansi supports further its close relationship to T. brucei.