A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the Nglycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic\apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3h to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-

Recently, it was shown that a cellulose-negative mutant (Cell) ofAcetobacterxylinum ATCC 23769 carried an insertion of an indigenous transposable element (IS1031A) about 500 bp upstream of the bcs operon, required for cellulose synthesis. Here we show that Cell can be complemented by wild-type DNA covering the insertion point. Nucleotide sequencing of this region revealed the presence of two open reading frames, ORF1 and ORF2. ORF2, which is disrupted by the IS1031A insertion in Cell, potentially encodes the complementing function. ORF1 encodes a protein (CMCax) with significant homology to previously described endoglucanases. A cloned DNA fragment containing ORF1 expressed a carboxymethyl cellulose-hydrolyzing activity in Escherichia coli. In A. xylinum, CMCax is secreted into the culture growth medium. The CMCax mature protein consists of 322 amino acids and has a molecular mass of 35.6 kDa.

Secretory nonpancreatic type II phospholipase A2 (snpPLA2) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA2 in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA2 in a variety of normal tissues suggests that snpPLA2 may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA2 localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA2 immunoreactivity was observed in the arterial media that was colocalized with alpha-actin-positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA2 was observed colocalized with CD68-positive macrophages and HHF-35-positive SMCs and extracellularly in the lipid core. snpPLA2 was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA2 had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA2: (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA2 was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA2 is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA2 can release proinflammatory lipids at places of LDL deposition in the arterial wall.

The Acetobacter xylinum gene (ce/B) encoding phosphoglucomutase (EC 5.4.2.2) has previously been cloned by complementation of cellulose-negative mutants. In the present report the nucleotide sequence of a 2.0 kb DNA fragment containing celB is described. Expression analysis using the bacteriophage T7 RNA polymerase promoter 410 resulted in identification of a probable translational start codon of celie, and this conclusion was confirmed by N-terminal amino acid sequencing of the recombinant protein. From the nucleotide sequence data it was deduced that cell3 encodes a protein with a calculated molecular mass of 59-6 kDa. A protein of similar size was visualized after in wifro transcription and translation, using the cloned 2.0 kb fragment as template. The results of an amino acid sequence comparison and a biochemical analysis indicated that the Cel6 protein is structurally and functionally related to the previously characterized human and rabbit phosphoglucomutases.