(4,5). AP remains in amyloid deposits for prolonged periods without protein catabolism (6) and might thereby contribute to the persistence which underlies their pathogenicity. However, the structure ofAP has not been well characterized. AP from only one individual has been completely sequenced at the protein level (7), and the two reports of the SAP cDNA sequence (8, 9), as well as preliminary reports of the glycostructure (10,11,37), are conflicting. We have therefore compared the complete covalent structures of SAP and AP, with particular emphasis on the oligosaccharide and its role in turnover of the SAP glycoprotein.MATERIALS AND METHODS Proteins. SAP was isolated from the serum (13) of two healthy subjects, from two large pools of normal serum containing 1-ml aliquots from each of >5000 different healthy volunteer blood donors, and from a pool of malignant effusion fluids containing material from >300 donors. AP was isolated from two spleens containing AA amyloid, two spleens and two livers with AL amyloid, one spleen containing [ArgW]apolipoprotein A-I amyloid, and the spleen, liver, heart, kidney, and adrenals of an individual patient with AA amyloidosis. Amyloidotic tissue was homogenized in 0.01 M Tris/0.14 M NaCl/0.01 M EDTA, pH 8.0, and the supernatant after centrifugation at 50,000 x g for 30 min was dialyzed extensively into 0.01 M Tris/0.14 M NaCl/0.002 M CaCl2, pH 8.0, before extraction of AP precisely as described for SAP from serum (13). All SAP/AP preparations were >991% pure in heavily overloaded SDS/polyacrylamide gradient gels (Excelgel, Pharmacia) run under reducing conditions and stained with brilliant blue R-350 or with silver.Glycan Structure Analysis. Oligosaccharides attached to SAP and AP were released by hydrazinolysis and radiolabeled with sodium borotritide at the reducing terminus, which incorporates a single label into each chain independently of its structure, so that the relative molar proportions of all glycans present can be estimated directly (14). These oligosaccharides were analyzed by high-voltage paper electrophoresis in pyridine/acetic acid water (3:1:387 by volume) at pH 5.4, 80 V/cm. They were then converted to neutral glycans by neuraminidase digestion and were analyzed on a highresolution 1.5 cm x 2 m Bio-Gel P-4 (<400 mesh) column by elution at 550C with water (14). The structure was determined by sequential exoglycosidase digestion in combination with permethylation and GS-MS (14). Heterogeneity of sialic acid linkage in the A-1 and A-2 structures was determined by digestion with neuraninidase from Newcastle disease virus, specific for a2-3 bonds, and neuraminidase from Arthrobacter ureafaciens, which cleaves a2-3(6) bonds, followed by selective exoglycosidase digestion and product analysis using permethylation and GC-MS (14). Oligosaccharide Modification. Bovine al acid glycoprotein (AGP) (Sigma) was desialylated by incubation at 800C for 1 hr at 2-5 mg/ml in 12.5 mM H2SO4 (16), resulting in the expected alteration in electrophoretic mobility in 1% agarose gel i...

The cellular sites of clearance and degradation of the pentraxin plasma proteins, C-reactive protein, the classical acute phase reactant, and serum amyloid P component (SAP), a universal constituent of amyloid deposits, were sought using the ligand 'MI-yramine cellobiose (TC) which is substantially retained within the cells in which catabolism takes place. Pentraxins labeled with '"I-TC showed the same in vitro and in vivo ligand binding and the same in vivo plasma to as the directly iodinated proteins and the native unlabeled pentraxins, indicating that their mode of clearance was likely to be physiological. After intravenous injection into mice and rabbits of human C-reactive protein, human SAP, and mouse SAP, each labeled with '"I-TC, most of the radioactivity remaining in the body at 24 h was located in hepatocytes. None was detected in other liver cells, and only traces were present in other viscera; the rest was in the carcass, representing intact pentraxins in the blood and extravascular compartment, and escaped label which had not yet been excreted. Hepatocytes are thus the single major site of pentraxin clearance and catabolism in vivo. This is consistent with the observation that SAP that has localized to amyloid deposits persists there and is not degraded. (J. CliU. Invest. 1994. 94:1390-1396

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Responses of principals in general practice and trainees to question asking when they would refer childreni with uninary tract infections Girls Boys