In this rat model of CsA-induced nephrotoxicity, renal insufficiency and characteristic histologic changes are associated with altered expression of matrix and matrix-regulating molecules. Based on our results with alpha-TGF-beta antibodies, many but not all of these nephrotoxic effects of CsA are mediated by TGF-beta.

SUMMARY Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, ␣-smooth muscle actin phenotype, and expression of ␤-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-␤ 1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, plateletand macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease. Thrombospondin-1 (TSP-1) and cellular fibronectin, particularly the alternatively spliced isoform containing an extradomain EIIIA (Fn-EIIIA), are highly expressed during embryogenesis (ffrench-Constant and

The last two steps in the de novo biosynthesis (20). Positive plaques were isolated through two rounds of selection. The UMP synthasespecific inserts were isolated from these positive recombinant plaques by EcoRI digestion and subcloned into the EcoRI site of pBR322 for further sizing and restriction map analysis. For isolation of UMP synthase genomic fragments, a human genomic library prepared in the A Charon 4A vector (21) was screened with the human UMP synthase-specific plasmid pHUSc22 (17) as described for the cDNA libraries.DNA Sequencing. Specific restriction fragments of the UMP synthase inserts were isolated from low-meltingtemperature agarose and subcloned into M13 cloningsequencing vectors mp8/mp9 (22) or mpl8/mpl9 (23). The sequence of the fragments was determined by the dideoxy Abbreviations: QDC, orotidine-5'-monophosphate decarboxylase;

I.

Edema Factor toxin (EF) of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3′,5′-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-Oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15 μg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin-(ET) catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.