Regulation of divalent metal transporter-1 by serine phosphorylation

Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake

Bioassay-guided fractionation to isolate compounds of onion (Allium cepa L.) affecting bone resorption

In this work a hydrophilic ethanolic extract of onion (Allium cepa L.) devoid of flavonoids was found to inhibit significantly bone resorption in vitro and in vivo, whereas the flavonoids, claimed to posses phytoestrogen-like properties, were devoid of activity in vivo and toxic in vitro. Thus, in order to isolate the bone resorption inhibitory constituent(s) of onion, the hydrophilic ethanolic onion extract was subjected to an in vitro bioassay-guided fractionation using (semi)-preparative chromatographic techniques. Biological activity, i.e. bone resorption inhibitory activity, was determined in vitro using the osteoclast resorption pit assay: Medium, containing the fraction under investigation, was added to osteoclasts settled on ivory slices. After a 24-hour incubation period osteoclasts were counted and the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits per osteoclasts and was compared to a negative control, i.e. medium only, and to calcitonin (10-12 M) as positive control. In this way, from the starting fraction which inhibited significantly (p < 0.05) the osteoclast activity at a dose of 30.0 mg / ml, a compound inhibiting significantly the osteoclast activity (0.53 mg / ml; p < 0.05) could be isolated. Structural analysis performed as well by nuclear magnetic resonance (NMR) as by electrospray-ionization mass-spectroscopy identified unambiguously the compound as γ-Lglutamyl-trans-S-1-propenyl-L-cysteine sulphoxide (γ-GPeCSO). Consequently, an adapted, scaled-up isolation by means of ion exchange - column chromatography was performed in order to isolate γ-GPeCSO in large amounts. Thus, sufficient amounts of γ-GPeCSO could be isolated to develop a high performance liquid chromatography method to quantify γ-GPeCSO in the fractions of the bioassay-guided fractionation. NMR experiments performed for structural confirmation of the isolated compound, revealed the presence of% acetic acid (weight/weight) in the sample which originated from solvents used in the ion exchange procedures. However, the acetic acid could be removed by solid phase extraction chromatography. Quantification of γ-GPeCSO in the fractions of the bioassay guided fractionation showed a significant (p < 0.05) correlation between the amounts of γ-GPeCSO therein and the osteoclast activity inhibition, thus indicating that γ-GPeCSO inhibited osteoclast activity in vitro

A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-guided fractionation inhibits resorption activity of osteoclasts

One gram of onion added to the food of rats inhibits significantly (p < 0.05) bone resorption as assessed by the urinary excretion of tritium released from bone of 9-week-old rats prelabeled with tritiated tetracycline from weeks 1 to 6. To isolate and identify the bone resorption inhibiting compound from onion, onion powder was extracted and the extract fractionated by column chromatography and medium-pressure liquid chromatography. A single active peak was finally obtained by semipreparative high-performance liquid chromatography. The biological activity of the various fractions was tested in vitro on the activity of osteoclasts to form resorption pits on a mineralized substrate. Medium, containing the various fractions or the pure compound, was added to osteoclasts of new-born rats settled on ivory slices. After 24 h of incubation, the tartrate-resistant acid phosphatase positive multinucleated cells, that is, osteoclasts, were counted. Subsequently, the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits/osteoclasts and was compared to a negative control, that is, medium containing 10% fetal bovine serum only and to calcitonin (10(-12) M) as a positive control. Finally, a single peak inhibited osteoclast activity significantly (p < 0.05). The structure of this compound was elucidated with high-performance liquid chromatography-electrospray ionization-mass spectrometry, time-of-flight electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The single peak was identified as gamma-L-glutamyl-trans-S-1-propenyl-L-cysteine sulfoxide (GPCS). It has a molecular mass of 306 Da and inhibits dose-dependently the resorption activity of osteoclasts, the minimal effective dose being approximately 2 mM. As no other peak displayed inhibitory activity, it likely is responsible for the effect of onion on bone resorption