The type III transforming growth factor ␤ (TGF␤) receptor (T␤RIII) binds both TGF␤ and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of T␤RIII signaling in vivo is not known. In this study, we have sought to determine the role of T␤RIII during development. We identified the predominant expression sites of ⌻␤RIII mRNA as liver and heart during midgestation and have disrupted the murine T␤RIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in ⌻␤RIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that T␤RIII is required during murine somatic development. To assess the effects of the absence of T␤RIII on the function of its ligands, primary fibroblasts were generated from T␤RIII-null and wild-type embryos. Our results indicate that T␤RIII deficiency differentially affects the activities of TGF␤ ligands. Notably, T␤RIII-null cells exhibited significantly reduced sensitivity to TGF␤2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that T␤RIII is an important modulator of TGF␤2 function in embryonic fibroblasts and that reduced sensitivity to TGF␤2 may underlie aspects of the T␤RIII mutant phenotype.Members of the transforming growth factor ␤ (TGF␤) family are potent regulators of multiple cellular functions, including cell proliferation, differentiation, migration, and death (35, 64). As such, the TGF␤s are critical regulators of the growth and morphogenesis of a variety of tissues. Three TGF␤ isoforms (TGF␤1 to -3) have been described in mammals and are encoded by distinct genes (36). Although the three ligands have similar biological activities in many in vitro assays, null mutations in the three genes result in mice with distinct phenotypes, suggesting that each ligand has a unique role during murine somatic development (14,42,50). In mammalian cells, the diverse actions of the TGF␤s are mediated by two distinct type I and type II serine/threonine kinase receptors (T␤RI and T␤RII, respectively), which are expressed on most cell types and tissues (35). T␤RI and T␤RII can form a latent receptor complex, and ligand binding is required for the activation of the receptor complex (65). Upon TGF␤ binding, the receptors rotate relatively within the complex (65, 66), resulting in phosphorylation and activation of T␤RI by the constitutively active and autophosphorylated T␤RII (62). The activated T␤RI then directly signals to downstream intracellular substrates, e.g., Smads (21, 61).Many other cell surface receptors have been identified (64). Among them is the type III TGF␤ receptor T␤RIII, which binds to all three TGF␤s (32). In contrast to the type I and II receptors, T␤RIII, also known as betaglycan, appears dispensable for TGF␤-mediated signal transduction since most cells that lack functional T␤RIII still respond to TGF␤ (8). The murine form of T␤RIII is an 850-...

(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...

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.