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The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.Iron is a trace element that is essential for life, since it plays a critical role in many biochemical and physiological mechanisms. As a consequence, nature has developed an array of elaborate processes for the absorption, storage, and transport of iron within the body, and a homeostatic balance between these mechanisms is essential for good health.The vast majority of dietary non-heme iron is absorbed in the duodenum, where it is most soluble due to the acidic environment. Bioavailable iron is always in the ferrous (Fe 2ϩ ) state, but most ingested iron is in the ferric (Fe 3ϩ ) form. Reduction of Fe 3ϩ to Fe 2ϩ can be promoted by the action of a putative ferric reductase (1) and reducing components in the diet, such as ascorbate (2). The regulation of iron absorption from the diet by the small intestine is of crucial importance in determining body iron status, and consequently, a great deal of research interest has focused on the cellular mechanisms involved in iron accumulation. This has culminated in the expression cloning of an iron transporter from rat duodenum (3) and the positional cloning of the human homologue, Nramp2 (4). In the Caco-2 cell model of human small intestinal enterocytes Nramp2 mRNA is down-regulated by increasing cellular iron status (5), suggesting a role in iron homeostasis. In addition, in both microcytic anemic (mk) mice (4) and Belgrade rats (6), there is a defect in intestinal iron transport that has been mapped to the gene encoding the protein Nramp2. Taken together these data suggest to us that Nramp2 should be expressed on human enterocyte plasma membranes and function as an iron transporter. Our present study has tested this hypothesis, and accordingly, we present evidence that human enterocytes absorb iron across their apical membrane in a pH-dependent fashion. Using cells loaded with the fluorescent dye BCECF, 1 we show that there is a concurrent intracellular acidification induced by iron in the bathing medium. With an antibody generated against Nramp2, we demonstrate the expression of a 66-kDa apical membraneresident p...

The effect of the putative iron regulatory peptide hepcidin on iron absorption was investigated in mice. Hepcidin peptide was synthesized and injected into mice for up to 3 days, and in vivo iron absorption was measured with tied-off segments of duodenum. Liver hepcidin expression was measured by reverse transcriptasepolymerase chain reaction. Hepcidin significantly reduced mucosal iron uptake and transfer to the carcass at doses of at least 10 g/mouse per day, the reduction in transfer to the carcass being proportional to the reduction in iron uptake. Synthetic hepcidin injections down-regulated endogenous liver hepcidin expression excluding the possibility that synthetic hepcidin was functioning by a secondary induction of endogenous hepcidin. The effect of hepcidin was significant at least 24 hours after injection of hepcidin. Liver iron stores and hemoglobin levels were unaffected by hepcidin injection. Similar effects of hepcidin on iron absorption were seen in irondeficient and Hfe knockout mice. Hepcidin inhibited the uptake step of duodenal iron absorption but did not affect the proportion of iron transferred to the circulation. The effect was independent of iron status of mice and did not require Hfe gene product. The data support a key role for hepcidin in the regulation of intestinal iron uptake. IntroductionRecent advances in molecular-level understanding of iron absorption regulation have implicated several genes as regulators of iron absorption, 2 of which (Hfe and hepcidin) have received particular attention. [1][2][3] Hepcidin was originally identified as an antimicrobial peptide synthesized in liver, but evidence from knockout mice suggests this peptide is a negative regulator of iron absorption. 3,4 Initial work implicated hepcidin as the long-sought "stores regulator" of iron absorption proposed by Finch. 5 However, recent work has suggested a wider role for this peptide as it also shows an expression pattern consistent with the "erythroid regulator." 6 A mutation in hepcidin has recently been implicated as a cause of juvenile hemochromatosis. 7 Transgenic mice overexpressing hepcidin were found to develop an iron-deficient phenotype, consistent with an effect on placental iron transport and intestinal iron absorption. 8 Frazer et al 9 provided data that quantitatively relates hepcidin expression to iron absorption rates and expression of duodenal transporters in an iron-deficient rat model. It can be deduced that a similar inverse correlation between hepcidin expression and iron absorption probably exists in humans, based on data provided by Nemeth et al 10 relating urinary hepcidin to serum ferritin levels. Thus far, however, no data measuring the direct effect of injecting hepcidin on iron absorption rates is available. We therefore synthesized hepcidin peptide and injected this into normal, iron-deficient, and Hfe knockout mice and measured iron absorption rates. Materials and methodsMice (129/Ola-C57BL/6 mixed background strain) with a 2-kb pgk-neor gene flanked by loxP sites replacing a...

The ischemic stroke is the third leading cause of death in developed countries. The C-terminal peptide of mechano-growth factor (MGF), an alternatively spliced variant of insulin-like growth factor 1 (IGF-1), was found to function independently from the rest of the molecule and showed a neuroprotective effect in vivo and in vitro. In vivo, in a gerbil model of transient brain ischemia, treatment with the synthetic MGF C-terminal peptide provided very significant protection to the vulnerable neurons. In the same model, ischemia evoked increased expression of endogenous MGF in the ischemia-resistant hippocampal neurons, suggesting that the endogenous MGF might have an important neuroprotective function. In an in vitro organotypic hippocampal culture model of neurodegeneration, the synthetic peptide was as potent as the full-length IGF-1 while its effect lasted significantly longer than that of recombinant IGF-1. While two peptides showed an additive effect, the neuroprotective action of the C-terminal MGF was independent from the IGF-1 receptor, indicating a new mode of action for this molecule. Although MGF is known for its regenerative capability in skeletal muscle, our findings demonstrate for the first time a neuroprotective role against ischemia for this specific IGF-1 isoform. Therefore, the C-terminal MGF peptide has a potential to be developed into a therapeutic modality for the prevention of neuronal damage.

Fluorescence nanocrystals or quantum dots (QDs) are engineered nanoparticles (NP) that have shown great promise with potential for many biological and biomedical applications, especially in drug delivery/activation and cellular imaging. The use of nanotechnology in medicine directed to drug delivery is set to expand in the coming years. However, it is unclear whether QDs, which are defined as NPs rather than small molecules, can specifically and effectively deliver drugs to molecular targets at subcellular levels. When QDs are linked to suitable ligands that are site specific, it has been shown to be brighter and photostable when compared with organic dyes. Interestingly, pharmaceutical sciences are exploiting NPs to minimize toxicity and undesirable side effects of drugs. The unforeseen hazardous properties of the carrier NPs themselves have given rise to some concern in a clinical setting. The kind of hazards encountered with this new nanotechnology materials are complex compared with conventional limitations created by traditional delivery systems. The development of cadmium-derived QDs shows great potential for treatment and diagnosis of cancer and site-directed delivery by virtue of their size-tunable fluorescence and with highly customizable surface for directing their bioactivity and targeting. However, data regarding the pharmacokinetic and toxicology studies require further investigation and development, and it poses great difficulties to ascertain the risks associated with this new technology. Additionally, nanotechnology also displays yet another inherent risk for toxic cadmium, which will enter as a new form of hazard in the biomedical field. This review will look at cadmium-derived QDs and discuss their future and their possible toxicities in a disease situation.

Ghrelin is a 28-residue peptide hormone that is principally released from the stomach during fasting and prior to eating. Two forms are present in human plasma: the unmodified peptide and a less abundant acylated version, in which octanoic acid is attached to the third residue, a serine, via an ester linkage. The acylated form of ghrelin acts as a ligand for the growth hormone secretagogue receptor and can stimulate the release of growth hormone from the pituitary gland. It also initiates behavioral and metabolic adaptations to fasting. Here we show that an immobilized form of ghrelin specifically binds a species of high density lipoprotein associated with the plasma esterase, paraoxonase, and clusterin. Both free ghrelin and paraoxon, a substrate for paraoxonase, can inhibit this interaction. An endogenous species of ghrelin is found to co-purify with high density lipoprotein during density gradient centrifugation and subsequent gel filtration. This interaction links the orexigenic peptide hormone ghrelin to lipid transport and metabolism. Furthermore, the interaction of the esterified hormone ghrelin with a species of HDL containing an esterase suggests a possible mechanism for the conversion of ghrelin to des-acyl ghrelin.Ghrelin is a peptide hormone that was purified from the stomach that can cause the release of growth hormone from the anterior pituitary gland (1). It has subsequently been found that ghrelin is predominantly released from the stomach prior to feeding (2), although other tissues have been shown to express the gene as well (3). Peripheral injections of ghrelin have been shown to increase feeding in both rats (4) and humans (5), and a course of injections leads to increased obesity in rats (4). Therefore, ghrelin can be seen as an important link between the stomach, appetite, and metabolism as well as playing a role in growth hormone release.The aim of this study was to establish whether ghrelin interacts with any other component of plasma. It was hypothesized that any interaction might be an important factor in determining the activity or longevity of ghrelin in plasma. Furthermore, an interaction in the plasma might be involved in the creation of the two distinct forms of ghrelin (6): the acylated form, in which octanoic acid is covalently bound to the peptide, and the more prevalent des-acyl form in which the peptide is unmodified. The acylated form of ghrelin stimulates the release of growth hormone from the pituitary gland, therefore any mechanism that might convert one form of ghrelin into the other could be an important factor in controlling the activity of ghrelin. MATERIALS AND METHODSA peptide corresponding to mature human ghrelin, with a cysteine residue substituted for the serine residue at the third position (S3C ghrelin), was synthesized by Fmoc 1 chemistry using a Rainin PS3 automatic peptide synthesizer (Protein Technologies). The peptide was purified to over 90% homogeneity by reverse-phase chromatography using a Varian 500LC HPLC. The molecular weight of the purified peptide ...

We investigated the effects of the iron regulatory peptide hepcidin on iron transport by the human intestinal epithelial Caco-2 cell line. Caco-2 cells were exposed to hepcidin for 24 hours prior to the measurement of both iron transport and transporter protein and mRNA expression. Incubation with hepcidin significantly decreased apical iron uptake by Caco-2 cells. This was accompanied by a decrease in both the protein and the mRNA expression of the iron-response element containing variant of the divalent metal transporter (DMT1[؉IRE]). In contrast, iron efflux and iron-regulated gene1 (IREG1) expression were unaffected by hepcidin. Hepcidin interacts directly with a model intestinal epithelium. The primary effect of this regulatory peptide is to modulate the apical membrane uptake machinery, thereby controlling the amount of iron absorbed from the

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.