"I was like intoxicated with this positivity": the politics of hope amongst participants in a trial of a novel spinal cord injury rehabilitation technology in South Africa.

PURPOSE: This study discussed the reports by participants in a randomised controlled trial of a novel intervention for spinal cord injury (SCI) rehabilitation in Cape Town, South Africa. MATERIALS AND METHODS: Sixteen participants were randomised to rehabilitation involving the use of robotic locomotor training, a novel technology, or to a group receiving an activity-based intervention. All participants were interviewed before the intervention and at six months follow-up. RESULTS: In a context in which rehabilitation services for SCI are virtually non-existent, all participants approached the study with enthusiasm and expressed gratitude for participation. They had high hopes for what the programme could achieve, with many believing, perhaps incorrectly, that the programme would help them walk independently again. While hope and enthusiasm are useful for adherence to experimental intervention studies, there is a danger, especially in poorly resourced contexts, for participants to experience considerable disappointment following false hope not being realised. This raises important ethical issues for researchers interested in the potential of new technologies to promote health in poorly resourced contexts. CONCLUSIONS: For clinicians, the path between supporting positive emotions (which may lead to positive outcomes), and confronting unrealistic hope (which may lead to negative outcomes) may be difficult. Follow-up with participants after re-integration into their communities is important to determine long-term psychological impact.Pan African Clinical Trial Number: PACTR201608001647143IMPLICATIONS FOR REHABILITATIONIn low-resource contexts where there is a low level of access to rehabilitation services, such access in the context of a trial of a new intervention may engender hope in a group of people with spinal cord injury. This hope may increase when a new technology is used, as was the case in this study.Hope can be very helpful to people entering rehabilitation, but unrealistic hope and expectations may have negative implications in the longer term.In this study, expectations of participants centred, unrealistically, around regaining the ability to walk again, despite past experiences and medical advice suggesting otherwise.A thin line exists between supporting high expectations and confronting unrealistic hope. This conundrum is difficult for the clinician, as both inappropriate hope and undue pessimism about an intervention have the potential to cause harm.Participant follow-up after the end of any innovative trial is important, not just to monitor physical progress, but also, where necessary, to support participants through a potential period of disillusionment when they find their expectations have not been fully met.

The acute phase protein lactoferrin is a key feature of Alzheimer's disease and predictor of Aß burden through induction of APP amyloidogenic processing.

Amyloidogenic processing of the amyloid precursor protein (APP) forms the amyloid-ß peptide (Aß) component of pathognomonic extracellular plaques of AD. Additional early cortical changes in AD include neuroinflammation and elevated iron levels. Activation of the innate immune system in the brain is a neuroprotective response to infection; however, persistent neuroinflammation is linked to AD neuropathology by uncertain mechanisms. Non-parametric machine learning analysis on transcriptomic data from a large neuropathologically characterised patient cohort revealed the acute phase protein lactoferrin (Lf) as the key predictor of amyloid pathology. In vitro studies showed that an interaction between APP and the iron-bound form of Lf secreted from activated microglia diverted neuronal APP endocytosis from the canonical clathrin-dependent pathway to one requiring ADP ribosylation factor 6 trafficking. By rerouting APP recycling to the Rab11-positive compartment for amyloidogenic processing, Lf dramatically increased neuronal Aß production. Lf emerges as a novel pharmacological target for AD that not only modulates APP processing but provides a link between Aß production, neuroinflammation and iron dysregulation.

Robotic Locomotor Training Leads to Cardiovascular Changes in Individuals With Incomplete Spinal Cord Injury Over a 24-Week Rehabilitation Period: A Randomized Controlled Pilot Study.

OBJECTIVE: To describe the effect of robotic locomotor training (RLT) and activity-based training (ABT) on cardiovascular indices during various physiological positions in individuals with spinal cord injury. DESIGN: Randomized controlled pilot study. SETTING: Private practice: Therapy & Beyond Centre - Walking with Brandon Foundation, Sports Science Institute of South Africa, Cape Town, South Africa. PARTICIPANTS: Participants with chronic traumatic motor incomplete tetraplegia (N=16) who resided in the Western Cape, South Africa. INTERVENTION: Robotic locomotor training (Ekso GT) and activity-based training over a 24-week intervention. MAIN OUTCOME MEASURES: Brachial and ankle blood pressure, heart rate, heart rate variability, and cardiovascular efficiency during 4 physiological positions. RESULTS: No differences between groups or over time were evident in resting systolic and diastolic blood pressure, ankle systolic pressure, ankle brachial pressure index, and heart rate variability. Standing heart rate at 24 weeks was significantly higher in the ABT group (95.58±12.61 beats/min) compared with the RLT group (75.14±14.96 beats/min) (P=.05). In the RLT group, no significant changes in heart rate variability (standard deviation R-R interval and root mean square of successive differences) was found between the standing and 6-minute walk test physiological positions throughout the intervention. Cardiovascular efficiency in the RLT group during the 6-minute walk test improved from 11.1±2.6 at baseline to 7.5±2.8 beats per meter walked at 6 weeks and was maintained from 6 to 24 weeks. CONCLUSIONS: Large effect sizes and significant differences between groups found in this pilot study support the clinical effectiveness of RLT and ABT for changing cardiovascular indices as early as 6 weeks and up to 24 weeks of rehabilitation. RLT may be more effective than ABT in improving cardiac responses to orthostatic stress. Based on heart rate variability metrics, the stimulus of standing has comparable effects to RLT on the parasympathetic nervous system. Cardiovascular efficiency of exoskeleton walking improved, particularly over the first 6 weeks. Both the RLT and ABT interventions were limited in their effect on brachial and ankle blood pressure. A randomized controlled trial with a larger sample size is warranted to further examine these findings.

Lactoferrin in Bone Tissue Regeneration.

Among the multiple properties exhibited by lactoferrin (Lf), its involvement in bone regeneration processes is of great interest at the present time. A series of in vitro and in vivo studies have revealed the ability of Lf to promote survival, proliferation and differentiation of osteoblast cells and to inhibit bone resorption mediated by osteoclasts. Although the mechanism underlying the action of Lf in bone cells is still not fully elucidated, it has been shown that its mode of action leading to the survival of osteoblasts is complemented by its mitogenic effect. Activation of several signalling pathways and gene expression, in an LRPdependent or independent manner, has been identified. Unlike the effects on osteoblasts, the action on osteoclasts is different, with Lf leading to a total arrest of osteoclastogenesis. Due to the positive effect of Lf on osteoblasts, the potential use of Lf alone or in combination with different biologically active compounds in bone tissue regeneration and the treatment of bone diseases is of great interest. Since the bioavailability of Lf in vivo is poor, a nanotechnology- based strategy to improve the biological properties of Lf was developed. The investigated formulations include incorporation of Lf into collagen membranes, gelatin hydrogel, liposomes, loading onto nanofibers, porous microspheres, or coating onto silica/titan based implants. Lf has also been coupled with other biologically active compounds such as biomimetic hydroxyapatite, in order to improve the efficacy of biomaterials used in the regulation of bone homeostasis. This review aims to provide an up-to-date review of research on the involvement of Lf in bone growth and healing and on its use as a potential therapeutic factor in bone tissue regeneration.

Human Mesenchymal Stem Cell Response to Lactoferrin-based Composite Coatings.

The potential of mesenchymal stem cells (MSCs) for implantology and cell-based therapy represents one of the major ongoing research subjects within the last decades. In bone regeneration applications, the various environmental factors including bioactive compounds such as growth factors, chemicals and physical characteristics of biointerfaces are the key factors in controlling and regulating osteogenic differentiation from MSCs. In our study, we have investigated the influence of Lactoferrin (Lf) and Hydroxyapatite (HA) embedded within a biodegradable PEG-PCL copolymer on the osteogenic fate of MSCs, previous studies revealing an anti-inflammatory potential of the coating and osteogenic differentiation of murine pre-osteoblast cells. The copolymer matrix was obtained by the Matrix Assisted Pulsed Laser Evaporation technique (MAPLE) and the composite layers containing the bioactive compounds (Lf, HA, and Lf-HA) were characterised by Scanning Electron Microscopy and Atomic Force Microscopy. Energy-dispersive X-ray spectroscopy contact angle and surface energy of the analysed coatings were also measured. The characteristics of the composite surfaces were correlated with the viability, proliferation, and morphology of human MSCs (hMSCs) cultured on the developed coatings. All surfaces were found not to exhibit toxicity, as confirmed by the LIVE/DEAD assay. The Lf-HA composite exhibited an increase in osteogenic differentiation of hMSCs, results supported by alkaline phosphatase and mineralisation assays. This is the first report of the capacity of biodegradable composite layers containing Lf to induce osteogenic differentiation from hMSCs, a property revealing its potential for application in bone regeneration.

Revisiting binding of plutonium to transferrin by CE-ICP-MS.

Capillary electrophoresis coupled with an inductively coupled plasma mass spectrometer was applied for the first time to determine the binding constant of human transferrin (Tf) for tetravalent plutonium. The experiments were carried out in a buffer 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6, 0.1 M NaCl and at a temperature of 25 °C. The nitrilotriacetate anion (NTA) used in this study prevents the hydrolysis of plutonium and is an ideal competitor with Tf for Pu, both ligands sharing comparable binding strength. The separation revealed unambiguous two peaks associated with the complex Pu(NTA)2 used as the initial species and with Pu-transferrin. Two series of independent experiments were conducted and gave the first stepwise conditional bicarbonate-free Pu-transferrin binding constant of . In the absence of bicarbonate the affinity of transferrin for plutonium at pH 6 is about 104 times stronger than that of iron at pH 6.7 .

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells.

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2, expressing iron-response-element-independent TFRC mRNA to promote cellular iron-overload and examined the effect of excess holotransferrin (5g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1, HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell-surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin-supplemented conditions for 24h and 48h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4h of treatment, while HFE expression altered later at 24h and 48h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

The importance of the general practitioner as an information source for patients with hereditary haemochromatosis.

OBJECTIVE: To explore hereditary haemochromatosis (HH) patients' perspectives on genetic information, namely the types of sources used, preferred or trusted. METHODS: A survey online was conducted by the European Federation of Associations of Patients with Haemochromatosis (EFAPH) and applied to members of nine National Associations. RESULTS: From a total of 1019 validated questionnaires, 895 respondents had performed a genetic testing for HH. From these, 627 self-declared that they were sufficiently informed about the implications of the genetic test to their health. The majority (66%) obtained the information from a specialist doctor, but would like to obtain it from the family doctor. However, the specialist was still the one they trusted more (69%). Regarding the 298 respondents who did not feel sufficiently informed, the majority (78%) also would like to have information from the family doctor although they also trusted the specialist more (75%). A different perspective was reported when patients were asked about the implications of the genetic testing to their family members, where the majority of respondents preferred obtaining information from a specialist (69%). CONCLUSION: This study elucidates the patients' needs for information and identifies the general practitioner (GP) as the preferred source to obtain information about HH. PRACTICE IMPLICATIONS: These results may have important implications in future strategies for HH awareness, giving a special emphasis on GPs as the main players.

MAPLE-based method to obtain biodegradable hybrid polymeric thin films with embedded antitumoral agents.

In this work, antitumor compounds, lactoferrin [recombinant iron-free (Apo-rLf)], cisplatin (Cis) or their combination were embedded within a biodegradable polycaprolactone (PCL) polymer thin film, by a modified approach of a laser-based technique, matrix-assisted pulsed laser evaporation (MAPLE). The structural and morphological properties of the deposited hybrid films were analyzed by Fourier-transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). The in vitro effect on the cells' morphology and proliferation of murine melanoma B16-F10 cells was investigated and correlated with the films' surface chemistry and topography. Biological assays revealed decreased viability and proliferation, lower adherence, and morphological modifications in the case of melanoma cells cultured on both Apo-rLf and Cis thin films. The antitumor effect was enhanced by deposition of Apo-rLf with Cis within the same film. The unique capability of the new approach, based on MAPLE, to embed antitumor active factors within a biodegradable matrix for obtaining novel biodegradable hybrid platform with increased antitumor efficiency has been demonstrated.

Characterization of the anti-HBV activity of HLP1-23, a human lactoferrin-derived peptide.

Lactoferrin (Lf) was shown to exhibit its antiviral activity at an early phase of viral infection and a mechanism whereby the protein interacts with host cell surface molecules has been suggested. In this study, human Lf (HLf) and seven HLf-derived synthetic peptides (HLP) corresponding to the N-terminal domain of the native protein (1-47 amino acids sequence) were assayed for their capacity to prevent hepatitis B virus (HBV) infection and replication using the HepaRG and HepG2.2.2.15 cell lines. Of the series tested, four peptides showed 40-75% inhibition of HBV infection in HepaRG cells, HLP1-23 , containing the GRRRR cationic cluster, being the most potent. Interestingly, this cluster is one of the two glycosaminoglycan binding sites of the native HLf involved in its antiviral activity; however, the mechanism of the HLP1-23 action was different from that of the full-length protein, the peptide inhibiting HBV infection when pre-incubated with the virus, while no effect was observed on the target cells. It is suggested that the cationic cluster is sufficient for the peptide to interact stably with negatively charged residues on the virion envelope, while the absence of the second glycosaminoglycan binding site prevents its efficient attachment to the cells. In conclusion, this peptide may constitute a non-toxic approach for potential clinical applications in inhibiting HBV entry by neutralizing the viral particles.

Structural basis for iron piracy by pathogenic Neisseria.

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

Iron mobilization from transferrin by therapeutic iron chelating agents.

BACKGROUND: The bacteriostatic activity of the transferrin family has been known since the early 1960's. The possession of high affinity iron(III)-binding sites and the existence of a specific membrane-bound receptor, have led to the present understanding of serum transferrin acting as the major iron transporter between cells in vertebrate systems. Iron chelators can interact with transferrin, either by directly donating iron or by removing iron from the protein; both interactions have relevance for haematology. SCOPE OF REVIEW: Urea polyacrylamide gels and HPLC methods have been developed for the resolution and quantification of the four major forms of transferrin, diferric-transferrin, C-mono Fe-transferrin, N-mono Fe-transferrin and apo transferrin. MAJOR CONCLUSIONS: Negatively charged ligands with pFe values >20 remove iron from transferrin, preferably from the N-lobe iron-binding site. Some siderophores are capable of removing iron from transferrin. 3-Hydroxypyridin-4-ones, lacking a negative charge are able to remove iron from transferrin with a strong preference for the C- lobe iron-binding site. The donation of iron to apo transferrin by hydroxypyridinone iron(III) complexes has relevance to the treatment of clinical anaemias, because the hydroxypyridinones can also mobilize iron from the reticuloendothelial system and so facilitate the redistribution of iron from macrophages to reticulocytes. GENERAL SIGNIFICANCE: Hydroxypyridinones have excellent potential for facilitating the redistribution of iron and this has relevance to the treatment of many disease types, including neurodegeneration and clinical anaemias. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

The transfer of iron between ceruloplasmin and transferrins.

BACKGROUND: It is over 60years since the discovery and isolation of the serum ferroxidase ceruloplasmin. In that time much basic information about the protein has been elucidated including its catalytic and kinetic properties as an enzyme, expression, sequence and structure. The importance of its biological role is indicated in genetic diseases such as aceruloplasminemia where its function is lost through mutation. Despite this wealth of data, fundamental questions about its action remain unanswered and in this article we address the question of how ferric iron produced by the ferroxidase activity of ceruloplasmin could be taken up by transferrins or lactoferrins. METHODS: Overlapping peptide libraries for human ceruloplasmin have been probed with a number of different lactoferrins to identify putative lactoferrin-binding regions on human ceruloplasmin. Docking software, 3D-Garden, has been used to model the binding of human lactoferrin to human ceruloplasmin. RESULTS: Upon probing the human ceruloplasmin library with human lactoferrin, three predominantly acidic lactoferrin-binding peptides, located in domains 2, 5 and 6 of human ceruloplasmin, were identified. The docking software identified a complex such that the N-lobe of human apo-lactoferrin interacts with the catalytic ferroxidase centre on human ceruloplasmin. GENERAL SIGNIFICANCE: In vitro binding studies and molecular modelling indicate that lactoferrin can bind to ceruloplasmin such that a direct transfer of ferric iron between the two proteins is possible. A direct transfer of ferric iron from ceruloplasmin to lactoferrin would prevent both the formation of potentially toxic hydroxyl radicals and the utilization of iron by pathogenic bacteria.

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae.

BACKGROUND: Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. RESULTS: The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 µm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. CONCLUSIONS: A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells.

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family "zyklopen."

Transferrin receptor 2 is crucial for iron sensing in human hepatocytes.

Hepcidin expression in vivo is regulated in proportion to iron status (i.e., increased by iron loading and decreased in iron deficiency). However, in vitro studies with hepatoma cell lines often show an inverse relationship between iron status and hepcidin expression. Here, we investigated possible molecular mechanisms responsible for the differences in iron sensing between hepatoma cell lines and human primary hepatocytes. RNA was collected from primary human hepatocytes, and HepG2 and HuH7 hepatoma cells were treated with either transferrin-bound and non-transferrin-bound iron. Expression of hepcidin, transferrin receptor 2, HFE, and hemojuvelin were quantified by real-time PCR. Hepcidin expression was increased in primary human hepatocytes following 24-h exposure to holoferric transferrin. In contrast, hepcidin mRNA levels in hepatoma cells were decreased by transferrin. Hepcidin expression was positively correlated with transferrin receptor 2 mRNA levels in primary human hepatocytes. Compared with primary hepatocytes, transferrin receptor 2 expression was significantly lower in hepatoma cell lines; furthermore, there was no correlation between transferrin receptor 2 and hepcidin mRNA levels in either HepG2 or HuH7 cells. Taken together our data suggest that transferrin receptor 2 is a likely candidate to explain the differences in iron sensing between hepatoma cell lines and primary human hepatocytes.

Liposomalization of lactoferrin enhanced its anti-tumoral effects on melanoma cells.

A number of studies have reported the anti-tumoral activity of lactoferrin, a property mediated by a variety of mechanisms such as inhibitory effects on tumor cell growth, NK cell activation, and enhancement of apoptosis. Liposomes are known to be an efficient drug delivery system which can enhance the therapeutic potential of the encapsulated compounds. We have used positively charged liposomes composed of phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE), cholesterol (Chol) and stearylamine (SA) (6:1:2:1 M ratio) as a carrier system for bovine iron-free Lf (ApoBLf), and compared the in vitro effect of free and liposome-entrapped ApoBLf on the growth and morphology of murine melanoma B16-F10 cells. Liposomal formulation of ApoBLf was found to enhance the capacity of the protein to inhibit the cell proliferation by affecting cell cycle progression. The effect appeared to be due to the capacity of liposomes to increase the uptake of the protein and its accumulation into cells and probably to protect it from degradation, as revealed by fluorescence microscopy and flow cytometry. Our results demonstrate the ability of liposomes to improve the anti-tumor activity of Lf and suggest that liposomal protein may have a potential therapeutic use in the prevention and/or treatment of cancer diseases.

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins.

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

Identification of an iron-hepcidin complex.

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.

Isolation of lactoferrin from milk of different species: calorimetric and antimicrobial studies.

Lactoferrin (LF) is an iron-binding glycoprotein found in different biological fluids of mammals and in neutrophils. It has been proposed to be involved in many functions, including protection from pathogens. In this work, purification of lactoferrin using an ion-exchange chromatography (SP-Sepharose) was attempted for the milk of the following animals: sheep (Ovis aries), goat (Capra hircus), camel (Camelus bactrianus), alpaca (Lama pacos), elephant (Elephas maximus) and grey seal (Halichoerus grypus), as well as human (Homo sapiens). Lactoferrin was identified in all the milks apart from that from grey seal. The thermal stability of the purified lactoferrins, in their native and iron-saturated forms, was studied by differential scanning calorimetry (DSC). Maximum temperature, onset temperature and enthalpy change of denaturation were higher when lactoferrins were saturated with iron than in their native form, indicating an increase in the stability of the protein structure upon iron-binding. Human lactoferrin was found to be the most heat-resistant and the other lactoferrins presented different degrees of thermoresistance, that of elephant being the least resistant. The antimicrobial activity of the different isolated lactoferrins was investigated against Escherichia coli 0157:H7. The minimal inhibitory concentrations (MICs) were determined by measuring the absorbance at 620 nm. The minimum bactericidal concentrations (MBCs) were also measured and it was found that camel lactoferrin was the most active lactoferrin against E. coli 0157:H7, whereas alpaca and human lactoferrins were the least active.