Aortic medial amyloid is a form of localized amyloid that occurs in virtually all individuals older than 60 years. a 50-aa-long peptide, here called medin, that is positioned within the coagulation factor-like domain of lactadherin. Our result is supported by the specific labeling of aortic medial amyloid in light and electron microscopy with two rabbit antisera raised against two synthetic peptides corresponding to different parts of medin. By Rabbit Polyclonal to JAK1 using hybridization we have shown that lactadherin is expressed by aortic medial smooth muscle cells. Furthermore, one of the synthetic NU7026 distributor peptides forms amyloid-like fibrils (L-70 ultracentrifuge with Ti 55.2 rotor; Beckman) for 20 min. The pellet was rehomogenized in the same buffer, and the procedure was repeated at least three times. The resulting pellet was washed with distilled water, suspended in 30 ml of distilled water, and centrifuged at 100,000 for 20 min to minimize SDS content. The final pellet was transferred to a glass bottle and extracted with 30 ml of 1 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for 30 NU7026 distributor min (22C) on a shaker. Amyloid content was monitored throughout the procedure by staining smears of the homogenates and extracts on slides for 10 min in Congo red B solution (11). The HFIP extract was filtered through several layers of sterile gauze to remove coarse debris. The crude extract then was transferred to 1.5-ml Eppendorf tubes and dried in a vacuum centrifuge (SC 100; Savant). Each dried pellet was delipidated by tip sonication (Ultrasonics, Farmingdale, NY) in chloroform/methanol (2:1) (700 l) followed by vortexing after the addition of 400 l of distilled water. After centrifugation at 10,000 for 1 min, the aqueous supernatants were removed carefully. Methanol (500 l) was added and proteins were pelleted by centrifugation at 10,000 for 1 min. The supernatant, containing 2C5 mg of dissolved protein, was loaded onto a 10 300-mm Superose 12 HR column (Amersham Pharmacia) and developed with 70% formic acid at a flow rate of 200 l/min. The effluent NU7026 distributor was monitored at 280 nm. RP-HPLC. Superose 12 HR fractions were subjected to slot-blot analysis (not shown) with antiserum A172. Reactive fractions (3- to 30-kDa range) were pooled, dried by vacuum centrifugation, and suspended in 60% formic acid. After 1 min of centrifugation at 10,000 Hybridization. A pGEM vector with a 1.6-kb insert of cDNA NU7026 distributor [EST 05878 (17)] corresponding to the complete coding sequence NU7026 distributor for the milk fat globule protein gene was obtained from the American Type Culture Collection (ATCC number 84574, HIBAA24 construct). A 122-bp segment (coding for amino acid residues 106C146 of lactadherin) was cut out from the vector by using (GIBCO/BRL). Riboprobes were transcribed from the plasmids with a digoxigenin RNA-labeling kit (T7/SP6; Boehringer Mannheim) according to the manufacturers instructions. The sense probe was used as a negative control. The probe sequence was verified by DNA sequencing, using the T7 Sequenase 2.0 sequencing kit (Amersham). Sections were cut and placed on poly-l-lysine-treated slides under RNase-free conditions. hybridization was performed as described (18, 19). Synthetic Peptides and Antisera. Two C-terminally amidated peptides with the sequences RLDKQGNFNAWV and NFGSVQFV, corresponding to the N- and C-terminal parts of the amyloid component (lactadherin, positions 245C256 and 286C293, respectively), were synthesized as described (20). The synthetic peptides were linked to keyhole limpet hemocyanin and used as immunogens for antisera production (nos. A172 and A177) in two rabbits as described (21). ELISA. ELISA was performed as described (22). Light and Electron Microscopy. Tissue sections (6 m) were cut from the paraffin-embedded aortic tissue, stained with alkaline Congo red, and examined in a polarization microscope for green birefringence (11). For immunohistochemistry, sections were incubated with antisera diluted 1:200 in TBS,.