Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid that activates cells involved in inflammation. The biological activity of PAF depends on its structural features, namely an ether linkage at the sn-1 position and an acetate group at the sn-2 position. The actions of PAF are abolished by hydrolysis of the acetyl residue, a reaction catalysed by PAF acetylhydrolase. There are at least two forms of this enzyme--one intracellular and another that circulates in plasma and is likely to regulate inflammation. Here we report the molecular cloning and characterization of the human plasma PAF acetylhydrolase. The unique sequence contains a Gly-Xaa-Ser-Xaa-Gly motif commonly found in lipases. Recombinant PAF acetylhydrolase has the substrate specificity and lipoprotein association of the native enzyme, and blocks inflammation in vivo: it markedly decreases vascular leakage in pleurisy and paw oedema, suggesting that PAF acetylhydrolase might be a useful therapy for severe acute inflammation.

CENP-A, a centromere-specific 17-kDa protein, has histone-like properties. However, in contrast to the common somatic histones, CENP-A is quantitatively retained in bull spermatozoa, and we have exploited this fact to purify CENP-A to apparent homogeneity. Partial sequence analysis of the purified protein indicates that CENP-A is a distinctive gene product. Some CENP-A sequences are highly similar to regions of histone H3. Other segments of CENP-A are not related to H3 or any other histone. These unrelated segments are presumably involved in loaliing CENP-A to centromeric DNA or in centromere-specific functions of CENP-A..The centromeric region of the chromosome is responsible for its integration into the mitotic spindle and for its proper segregation poleward during anaphase (1)(2)(3)(4). Several proteins have been identified that are associated with the centromere (5-10). For the most part, their functions are presently unknown. Recently, however, Bischoff et al. (11) reported that a 47-kDa centromere-specific autoantigen is highly homologous to the translation product ofRCCI, a gene involved in regulating mammalian chromosome condensation. This antigen may be identical to CENP-D, a 50-to 60-kDa centromeric protein reported to be present in a variety of species (7, 9, 10).CENP-A, a centromere-specific protein of 17 kDa, appears to be associated with kinetochore chromatin or with chromatin closely apposed to the outermost domain of the kinetochore, as judged by indirect immunofluorescence (9) and immunoelectron microscopy (12). This localization suggests it may play a direct role in kinetochore function during mitosis.We have extensively analyzed CENP-A and have shown that it appears to be a core histone by the following criteria: (i) it is extracted with histones from chromatin by dilute mineral acids, (ii) it elutes with histones H3 and H4 in apparent tetrameric complexes during ion exchange (13) or sizing-column chromatography (D.K.P., K.O., and R.L.M., unpublished observations), and, most importantly, (iii) it is a component of highly purified nucleosome core particles (13). CENP-A is exceptional among somatic histones not only because it is centromere specific but also because it is quantitatively retained in chromatin during spermatogenesis in mammals, even in species where other histones are quantitatively replaced by protamines (14).The selective retention of this protein in bull sperm, where other histones are absent, has now allowed us to extract and purify CENP-A to homogeneity. We have also subjected the purified CENP-A from bull sperm to partial sequence analysis and report here that it is a distinctive histone, with sequences similar to those of H3, as well as segments that are not related to histones or homologous to any other known mammalian centromeric protein sequences (11, 15). MATERIALS AND METHODSPurification and Acid Extraction of Nuclei ad ReversePhase Chromatography of Acid-Extracted Proteins. Calf thymus nuclei and bull sperm nuclei were purified and extracted with 1 M NaCl/0.25...

The leukocyte-restricted beta 2 (CD18) integrins mediate cell adhesion in a variety of events essential for normal immune function. Despite extensive research in this field, only three members of this integrin subfamily have been described: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150,95). We have identified a cDNA encoding a fourth alpha chain, alpha d, that associates with CD18. The alpha d subunit is more closely related to CD11b and CD11c than to CD11a. This integrin is expressed at moderate levels on myelomonocytic cell lines and subsets of peripheral blood leukocytes, and more strongly on tissue-compartmentalized cells such as foam cells, specialized macrophages found in aortic fatty streaks that may develop into atherosclerotic lesions. The alpha d/CD18 molecule exhibits preferential recognition of ICAM-3 over ICAM-1.

Platelet-activating factor (PAF) is a potent pro-inflammatory autacoid with diverse physiological and pathological actions. These actions are modulated by PAF acetylhydrolase, which hydrolyzes the sn-2 ester bond to yield the biologically inactive lyso-PAF. In contrast to most secreted phospholipase A 2 s, plasma PAF acetylhydrolase is calcium-independent and contains a GXSXG motif that is characteristic of the neutral lipases and serine esterases. In this study we tested whether the serine in this motif is part of the active site of plasma PAF acetylhydrolase and, if so, what the other components of the active site are. Using site-directed mutagenesis, we demonstrated that Ser-273 (of the GXSXG motif), Asp-296, and His-351 are essential for catalysis. These residues were conserved in PAF acetylhydrolase sequences isolated from bovine, dog, mouse, and chicken. The linear orientation and spacing of these catalytic residues are consistent with the ␣/␤ hydrolase conformation of other lipases and esterases. In support of this model, analysis of systematic truncations of PAF acetylhydrolase revealed that deletions beyond 54 amino acids from the NH 2 terminus and 21 from the COOH terminus resulted in a loss of enzyme activity. These observations demonstrate that although plasma PAF acetylhydrolase is a phospholipase A 2 it has structural properties characteristic of the neutral lipases and esterases.

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD ؉ in a reaction that is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.Biochemical pathways that function in the recognition and repair of DNA damage are critical for maintaining genomic integrity. Potentially devastating DNA damage in the form of double-strand breaks (DSBs) 1 can occur when cells are exposed to ionizing radiation, oxidative stress and radiomimetic drugs. The DNA-dependent protein kinase (DNA-PK) is a key component of DNA DSB rejoining pathways in mammalian cells. DNA-PK is a heterotrimeric enzyme complex comprised of a 460-kDa catalytic subunit (1) and a regulatory component consisting of the Ku70 and Ku80 proteins (2, 3). The Ku70 and Ku80 proteins form a heterodimeric complex that binds to the ends of double-stranded DNA with high affinity (4 -8). The catalytic subunit of DNA-PK (DNA-PKcs) binds to the Ku70/80 complex in the presence of double-stranded DNA (9) and phosphorylates a wide variety of protein substrates in vitro on serine and threonine residues (10, 11). The protein kinase activity of DNA-PK is autoregulatory; in the absence of a phosphorylation substrate, DNA-PKcs autophosphorylates and dissociates from the Ku70/80-DNA complex (12).Evidence that DNA-PK plays an integral role in the repair of DNA DSB has been provided through the characterization of rodent cell lines that have mutations that disrupt the expression of the Ku80 (13-19) or DNA-PKcs (20 -24). Although it is clear that DNA-PK is an important component of mammalian DNA DSB repair pathways, it is not known how the enzyme participates in these processes. In vitro, DNA-PK preferentially phosphorylates protein substrates that co-localize on the same DNA molecule (3,25). This suggests that the specificity of the phosphorylation reaction may be regulated, in part, via the co-localization of the enzyme and substrate target on DNA. Based on these data, it has been proposed that DNA-PK could participate in the DNA rejoining reaction by phosphorylating DNA repair factors that co-localize with it on broken DNA en...