Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans.

The molybdenum cofactor (Moco) is a 520-Da prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy; however, the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a Celegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Rational design of protein-based MRI contrast agents.

We describe the rational design of a novel class of magnetic resonance imaging (MRI) contrast agents with engineered proteins (CAi.CD2, i = 1, 2,..., 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high-coordination Gd(3+) binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd(3+) over physiological metal ions such as Ca(2+), Zn(2+), and Mg(2+). These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small-molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, thereby extending applications of MRI.

Synthesis and screening of a library of Re/Tc-based amyloid probes derived from beta-breaker peptides.

Through the development and application of a unique approach for producing Re-metallopeptides, a new class of peptide-derived probes that are designed to target beta-amyloid plaques was developed. Derivatives of a class of beta-breaker peptides having the core sequence lvffa or affvl (lower case letters represent D-amino acids) and the single amino acid chelate quinoline (SAACQ) ligand which can bind Re and (99m)Tc were prepared on an automated peptide synthesizer. Both monomeric and dimeric peptides were synthesized in modest to good yields where in select examples a biotin-containing amino acid derivative was included to act as a linker point for further conjugation to carrier proteins. The Re complexes for all reported peptides were prepared similarly and screened for their ability to inhibit fibrillogenesis. Two of the reported compounds showed excellent inhibitory properties (8a: 40 +/- 5% amyloid formation versus control; 16: 40 +/- 4%) and warrant further investigation. For one of these leads, the (99m)Tc analogue was synthesized and the product showed high stability toward histidine and cysteine challenges, making it a viable candidate for in vivo biodistribution studies.

Biological activities of selected peptides: skin penetration ability of copper complexes with peptides.

This study concerning the permeability through skin barriers of copper complexes with peptides is an important part of the research on their biological activity. The transport of copper complexes through the skin is essential in treatment of dermatological dysfunctions connected to the deficiency of these elements in the skin. During the last several years, a special interest in transepidermal copper delivery has been observed. This is the reason why copper compounds have been used as active compounds in care cosmetics. Yet, the transport process of copper complexes with tripeptides, glycyl-histidyl-lysine GHK, or gamma-glutamyl-cysteinyl-glycine GSH through the stratum corneum has received very little attention in the literature so far. The penetration ability of GHK-Cu and GSH-Cu through the stratum corneum and the influence of the complexes with tripeptide on the copper ion transport process is the key factor in their cosmetic and pharmaceutical activity. The in vitro penetration process was studied in the model system, a Franz diffusion cell with a liposome membrane, where liquid crystalline systems of physicochemical properties similar to the ones of the intercellular cement of stratum corneum were used as a standard model of a skin barrier. The results obtained demonstrated that copper complexes permeate through the membranes modeling the horny lipid layer and showed the influence of peptides on the dynamics of copper ion diffusion.

The pathogenesis of molybdenum cofactor deficiency, its delay by maternal clearance, and its expression pattern in microarray analysis.

Molybdenum cofactor (Moco)-deficiency is a lethal autosomal recessive disease, for which until now no effective therapy is available. The biochemical hallmark of this disorder is the inactivity of the Moco-dependent sulfite oxidase, which results in elevated sulfite and diminished sulfate levels throughout the organism. In humans, Moco-deficiency results in neurological damage, which is apparent in untreatable seizures and various brain dysmorphisms. We have recently described a murine model for Moco-deficiency, which reflects all enzyme and metabolite changes observed in the patients, and an efficient therapy using a biosynthetic precursor of Moco has been established in this animal model. We now analyzed these mice in detail and excluded morphological brain damage, while expression analysis with microarrays indicates a massive cell death program. This neuronal damage appears to be triggered by elevated sulfite levels and is ameliorated in affected embryos by maternal clearance.

Involvement of iron (ferric) reduction in the iron absorption mechanism of a trivalent iron-protein complex (iron protein succinylate).

Iron protein succinylate is a non-toxic therapeutic iron compound. We set out to characterise the structure of this compound and investigate the importance of digestion and intestinal reduction in determining absorption of the compound. The structure of the compound was investigated by variable temperature Mössbauer spectroscopy, molecular size determinations and kinetics of iron release by chelators. Intestinal uptake was determined with radioactive compound force fed to mice. Reduction of the compound was determined by in vitro incubation with intestinal fragments. The compound was found to contain only ferric iron, present as small particles including sizes below 10 nm. The iron was released rapidly to chelators. Digestion with trypsin reduced the molecular size of the compound. Intestinal absorption of the compound was inhibited by a ferrous chelator (ferrozine), indicating that reduction to ferrous iron may be important for absorption. The native compound was a poor substrate for duodenal reduction activity, but digestion with pepsin, followed by pancreatin, released soluble iron complexes with an increased reduction rate. We conclude that iron protein succinylate is absorbed by a mechanism involving digestion to release soluble, available ferric species which may be reduced at the mucosal surface to provide ferrous iron for membrane transport into enterocytes.

Fate of cadmium bound to phytochelatin in rats.

The fate cadmium(Cd) bound to phytochelatin [PC, (gamma-Glu-Cys)n-Gly)] was studied in rats using synthesized 109Cd-PC. Less Cd was absorbed through the digestive tracts than CdCl2, but the ratio of renal Cd to hepatic Cd was higher. After parenteral administration of Cd-PC, Cd was distributed mainly in the liver, kidney, small intestine and pancreas. More Cd was found in the kidney than the liver after Cd-PC (n = 5) administration. Most of the Cd was bound to the high molecular weight fraction in the hepatic cytosol 0.5 hr after administration and moved to the metallothionein fraction at 6 hr. The tissue distribution of Cd was not affected even when free PC (n = 5) was administered 3 hr after or before Cd injection. The distribution in the kidney increased only in the case of the simultaneous administration of Cd with PC. These findings show that the absorbance of Cd bound to PC from the alimentary tract is lower than that of CdCl2 although absorbed Cd is distributed to the kidney more than CdCl2, and Cd is liberated from PC soon after uptake by the cells.

Absorption of iron from iron succinyl-protein complexes by mouse small intestine.

The absorption of iron from iron succinyl-protein complexes was investigated in mice. 59Fe-labelled succinyl-casein and -albumin complexes, [59Fe]ferritin and 59FeSO4, at doses of 20 or 200 micrograms of iron, were administered orally to normal mice or mice with absorption enhanced by chronic hypoxia. 59Fe from iron succinyl-protein was well absorbed in normal mice (greater than 10% of dose) and showed enhanced absorption in hypoxic mice (greater than 40% of dose). Intestinal uptake was predominantly by the duodenum for all compounds studied. In-vivo absorption of 59Fe from an iron succinyl-protein complex was studied using tied-off segments of mouse duodenum, jejunum or ileum of normal or hypoxic mice. Incubation for up to 15 min in duodenum or 60 min in ileum showed very little absorption of 59Fe. No enhancement of absorption was seen in hypoxic mice. It was concluded that absorption of the intact iron succinyl-protein complex cannot explain absorption seen after oral dosing.

Comparison of anti-anaemic effects of iron protein succinylate (ITF 282) and ferrous sulfate in the rat.

The effects of iron protein succinylate (ITF 282), an iron complex insoluble under acid conditions, and ferrous sulfate were studied in the rat. After a single oral administration of ITF 282, the concentration of free iron in the stomach was less than the 10% of that found after a corresponding dose of ferrous sulfate and the effect of induction of diarrhoea was significantly less important than that of ferrous sulfate. Iron absorption and kinetics were evaluated by measuring serum iron after oral administration of equidoses of iron to the anaemic rat. The results showed that ferrous sulfate induces higher serum iron levels than ITF 282. The anti-anaemic effects of the two iron products were studied during 4 weeks of treatment (3 mg iron/kg/day): the data obtained confirmed that ferrous sulfate is significantly more potent in increasing serum iron, but in contrast both compounds are equally active in restoring normal haemoglobin values.

The action of the collagen-Fe2+ complex at tissular level.

The aim of this paper is to know the iron dynamics and action of the collagen-Fe2+ complex at tissular level. The results showed that the collagen-Fe2+ complex is biocompatible at tissular level and the ferrous iron entered the animal organism on the well-known metabolic pathways. When a high dose of the complex was administered, an overloading with hemosiderin of macrophages and hepatocytes was noticed.