Haile, Audie Murphy Hospital, San Antonio, Texas) and anti–β-actin (1/10 000 clone AC-1 Sigma-Aldrich). The primary antibodies used were anti-ferroportin (1/2500 Dr D. Briefly, protein samples (100 μg) were separated on NuPAGE Bis-Tris 4% to 12%, 1.5 mm gels (Invitrogen) and then transferred to Invitrolon PVDF membranes. Western analysis was performed by established methods 34, 35 using the Invitrogen NuPAGE Novex System for optimal detection (Carlsbad, CA). J774 cells (ATCC) were grown using standard procedures 33 and used when at approximately 90% confluence to maximize ferroportin detection. Determination of the K d for hepcidin-α 2-M and hepcidin-α 2-M-MA We also verified that the hepcidin concentration in the control tubes was the same before and after centrifugation. In preliminary experiments using the ultracentrifugation of α 2-M and α 2-M-MA without any hepcidin added, we proved by measurement of total protein (Quick Start Protein Assay Kit Bio-Rad) that no α 2-M remained in the supernatant after ultracentrifugation, as it was completely pelleted. The number of hepcidin binding sites was calculated as the molar ratio of the bound hepcidin to total α 2-M. The amount of hepcidin bound to α 2-M or α 2-M-MA was calculated by subtracting the hepcidin concentration in the supernatant before and after ultracentrifugation. The mixture of α 2-M or α 2-M-MA with hepcidin (molar ratio, 1:5 at this molar ratio hepcidin is above the α 2-M and α 2-M-MA saturating concentration) was incubated at 37☌ for 2 hours and then subjected to ultracentrifugation at 180 000 g for 2 hours at 37☌. The demonstration that α 2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases. In fact, the α 2-M–hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. Because α 2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to α 2-M may influence its functions. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Surprisingly, the interaction of hepcidin with activated α 2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity ( K d 0.3 μM) hepcidin-binding sites. Hepcidin binding to nonactivated α 2-M displays high affinity ( K d 177 ± 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Interaction of 125I-hepcidin with α 2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. In this study, we identify α 2-macroglobulin (α 2-M) as the specific hepcidin-binding molecule in blood. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. Texts: Ojibwa, Lakota, Salish, Anishinabe, Eskimo, Quechua, Yokut, Navajo, Arapaho, Comanche and Jane Johnston Schoolcraft, W.W.Hepcidin is a major regulator of iron metabolism. more music: Jean-Christophe Rosaz words: North and South Amerindian poetry for soprano solo, choir and orchestra by Yolanda Fresedo (soprano), Ecce Cantus Choir and Orchestra conducted by Martina Niernhaussen in St Louis en l'Île church Paris Mai 22 2014 Music: Jean-Christophe Rosaz words: North and South Amerindian poetry for soprano solo, choir and orchestra by Yolanda Fresedo (soprano), Ecce Cantus Choir and Orchestra conducted by Martina Niernhaussen in St Louis en l'Île church Paris Mai 22 2014 Video: Indian Songs for soprano solo, choir and orchestra in eight parts
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