No abstract

Exposure of endothelial and many other cell types to tumor necrosis factor ␣ generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-B that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor ␣-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-B activity. Here we demonstrate that XIAP, a NF-Bdependent member of the IAP gene family, is a strong stimulator of NF-B. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-B via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.

The primary structure ofporcine brain (3-tubulin was determined by automated and manual Edman degradation of six sets of overlapping peptides. The protein consists of 445 amino acid residues and has a minimum of six positions that are heterogeneous, indicating at least two (3-tubulins in porcine brain. Com-parison of the optimally aligned sequences of a-tubulin and 13-tubulin indicates that 41% of their primary structures are identical. A region rich in glycyl residues is similar both in sequence and predicted secondary structure to the phosphate binding loop of several nucleotide binding enzymes. fi-Tubulin contains a highly acidic COOH-terminal region that resembles the NH2-terminus of troponin T.Microtubules are helical arrays of alternating globular units of a-tubulin and 83-tubulin, each having a molecular weight of 50,000, a similar amino acid composition, and a similar overall shape (for review, see ref. 1). Yet these subunits seem to differ in function, as indicated by the binding of exchangeable GTP to f3-tubulin (2) and by the participation of a tissue-specific /3-tubulin in the meiotic spindle and other stages of spermatogenesis in Drosophila (3, 4). P-Tubulin mutants have been shown to cause reduced binding of cytostatic drugs (5,6) and were used to demonstrate the involvement of microtubules in nuclear movement in germinating spores of Aspergillus (7). A knowledge of the primary structure of ,3-tubulin, along with that of a-tubulin, could be helpful in understanding how different tubulins function, how the assembly of microtubules into various organelles is regulated, and how certain drugs and nucleotides are bound with high affinity. The sequence of a-tubulin has already shown several variants of this protein in brain (8). Regional similarities were found to muscle proteins, indicating the possibility ofcommon biochemical features in muscular and microtubular movement. Here we present the sequence of l3-tubulin from porcine brain and compare it with that of the a-chain. MATERIALS AND METHODSTubulin was purified from porcine brain as described (8). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer, pH 7.0, was chromatographed on DEAE-cellulose with a linear gradient of0. 1-0. 3 M sodium chloride. The protein was reduced and alkylated with iodoacetic acid and assayed for contaminating proteins by disc gel electrophoresis in the system of Yang and Criddle (9) using 8% polyacrylamide gels. a-and ,/3chains were separated on hydroxylapatite in 0.1% NaDodSO4 with a linear gradient of 0.2-0.4 M sodium phosphate (10). Fractions were assayed for purity by gel electrophoresis as above. Only }3-chain preparations containing <5% impurities were used for sequence determination.To remove NaDodSO4, the protein was dialyzed against 1 mM ammonium bicarbonate, then, the solution was concentrated by vacuum evaporation and brought to pH 5.5 with acetic acid, and the protein was precipitated with 9 vol of ice-cold acetone. The supernatant was discarded after 2 hr at -200C, and the precipit...

Complementary DNA encoding human cyclophilin, a specific cyclosporin A‐binding protein, has been isolated from the leukemic T‐cell line Jurkat and sequenced. Comparison of the deduced amino acid sequence with the previously determined sequence of bovine thymus cyclophilin reveals only three differences: an additional amino acid at the carboxy terminus end and two internal changes. RNA transfer blot analysis indicates an mRNA size of approximately 1 kb for human T‐cell cyclophilin. Phytohaemagglutinin and phorbol myristate acetate induction of T cells treated or not with cyclosporin A affects only marginally the level of cyclophilin mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.

SummaryDuring the inflammatory response, endothelial cells (EC) transiently upregulate a set of genes encoding, among others, cell adhesion molecules and chemotactic cytokines that together mediate the interaction of the endothelium with cells of the immune system. Gene upregulation is mediated predominantly at the transcriptional level and in many cases involves the transcription factor nuclear factor (NF) KB. We have tested the concept of inhibiting the inflammatory response by overexpression of a specific inhibitor of NF-KB, IKBot. A recombinant adenovirus expressing Ir, B~x was constructed (rAd.IKBe 0 and used to infect EC of human and porcine origin. Ectopic expression of IgJ3a resulted in marked, and in some cases complete, reduction of the expression of several markers of EC activation, including vascular cell adhesion molecule 1, interleukins 1, 6, 8, and tissue factor. Overexpressed IKBot inhibited NF-K_B specifically since (a) in electrophoretic mobility shift assay, NF-r,B but not AP-1 binding activity was inhibited, and (b) von Willebrand factor and prostacyclin secretion that occur independently of NF-r,B, remained unaffected. Functional studies of leukocyte adhesion demonstrated strong inhibition of ilL-60 adhesion to IKBot-expressing EC. These findings suggest that NF-KB could be an attractive target for therapeutic intervention in a variety of inflammatory diseases, including xenograft rejection.