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Lysozymes are antibacterial effectors of the innate immune system in animals that hydrolyze peptidoglycan. Bacteria have evolved protective mechanisms that contribute to lysozyme tolerance such as the production of lysozyme inhibitors, but only inhibitors of chicken (c-) and invertebrate (i-) type lysozyme have been identified. We here report the discovery of a novel Escherichia coli inhibitor specific for goose (g-) type lysozymes, which we designate PliG (periplasmic lysozyme inhibitor of g-type lysozyme). Although it does not inhibit c- or i-type lysozymes, PliG shares a structural sequence motif with the previously described PliI and MliC/PliC lysozyme inhibitor families, suggesting a common ancestry and mode of action. Deletion of pliG increased the sensitivity of E. coli to g-type lysozyme. The existence of inhibitors against all major types of animal lysozyme and their contribution to lysozyme tolerance suggest that lysozyme inhibitors may play a role in bacterial interactions with animal hosts.

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The Atlantic salmon (Salmo salar) goose-type lysozyme gene was isolated and revealed alternative splicing within exon 2 affecting the signal peptide-encoding region. The lysozyme was produced in Escherichia coli, and the recombinant enzyme showed a high specific lytic activity that was stimulated by low or moderate concentrations of mono- or divalent cations. Relative lytic activities of 70 and 100% were measured at 4 degrees C and 22 degrees C, respectively, and there was no detectable activity at 60 degrees C. However, 30% activity was retained after heating the enzyme for 3 h at 90 degrees C. This unique combination of thermal properties was surprising since the salmon goose-type lysozyme contains no cysteines for protein structure stabilization through disulphide bond formation. The results point to a rapid reversal of inactivation, probably due to instant protein refolding.

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Genome clones and expressed sequence tags (ESTs) from the ascidian Ciona intestinalis and from the larvacean Oikopleura dioica were analysed for the presence of lysozyme-encoding genes. Two genes were found to potentially code for goose-type lysozymes in Oikopleura, while three or possibly more g-type proteins form the lysozyme complement of C. intestinalis, and at least one of these genes from each species is expressed based on EST data. No genes for chicken- or invertebrate-type lysozymes were found in either urochordate species. Consistent with this finding, extracts of Oikopleura animals possessed hydrolysing activity on bacterial cell walls, and this activity was not inhibited in the presence of a known inhibitor of chicken-type lysozyme. A wide range of isoelectric points for the predicted lysozymes from Ciona (pI 4.4, 6.4 and 9.9) and from Oikopleura (pI 5.0 and 8.0) suggests tissue-specific adaptations as well as specific functional roles of the lysozymes. Comparisons of gene structures, encoded sequences, cysteine residue content and their positions in the proteins indicate that the g-type lysozymes of Ciona intestinalis are more closely related to those of vertebrates than are the g-type lysozymes of Oikopleura. Multiple genes from each species may result from separate and lineage-specific duplications followed by functional specialisation.

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In a recent publication we reported the protein purification, characterization, and the gene isolation of a cDNA encoding the antibacterial cold-active lysozyme-like protein chlamysin from the marine bivalve Chlamys islandica. A 4.2 kb genomic chlamysin gene has now been amplified and sequence-analyzed. By comparison to the cDNA sequence and its translation product, the coding region was found separated in four exons of 38-252 bp. The introns range in size from 0.8 to 1.5 kb, and have traditional spliceosomal intron 5'-GT donor and 3'-AG acceptor sites for splicing. Two of the introns contain multiple copies of three sequence motifs not found repeated in other published genes. The over-all gene organization of chlamysin resembles chicken-type (c-type) lysozyme genes in vertebrates, but is different from the three-exon structure in invertebrate c-type lysozyme genes. A phylogenetic analysis of invertebrate-type (i-type) and c-type lysozyme proteins demonstrated a large evolutionary distance between the i-type and the c-type enzyme classes. Exons of the i-type genes are not equally organized according to their homolog protein domains.

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An antibacterial approximately 11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5-6.2) and at low temperature (4-35 degrees C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70 degrees C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.

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A crude lysozyme preparation was recovered in waste from the scallop processing industry. Lysozyme was then purified 229-fold in preparative scale by chromatography on S Sepharose and Blue Sepharose. Further purification on Sephacryl S-200 resulted in a lysozyme preparation with a specific activity of 64,000 units/mg protein. The apparent molecular mass of the partially purified lysozyme was 10 kDa as judged by gel filtration. Optimum pH for lysis of Micrococus luteus under the present conditions was 5.2. The enzyme was very active at low temperatures. At 4 degrees C the scallop viscera lysozyme exhibits about 55% of the activity measured at 37 degrees C.

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DNA repair capacity is likely to be a critical factor in mutagenesis and carcinogenesis, as well as for the response to some cytostatics. We have studied inter- and intra-individual variation in the activities of O6-methylguanine--DNA methyltransferase (O6-MT) and uracil--DNA glycosylase (UDG) in 35 placentae from smokers and non-smokers. The maximum interindividual variation in the activities of O6-MT and UDG were 8.3- and 7.7-fold, respectively. The corresponding intraindividual variations were 2.7- and 3.3-fold. Generally, a high level of O6-MT activity was accompanied by a high O6-MT mRNA level, but no such correlation was seen for UDG. These results were not due to degradation of the enzymes or mRNAs after delivery. No correlation between the activities of O6-MT and UDG was observed, indicating that they are differentially regulated. A 1.4-fold (P < or = 0.05) higher activity of O6-MT was observed in smokers as compared to non-smokers, indicating a small, but statistically significant difference. No significant difference was observed for UDG. Our results demonstrate that DNA repair capacities vary largely between different individuals, and that environmental factors may modulate the expression of DNA repair enzymes.

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Uracil-DNA glycosylase, the enzyme that catalyzes the release of free uracil from single-stranded and double-stranded DNA, has been purified 26,600-fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil-DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme. This report also presents a 700-fold purification of HeLa S3 cell O6-methylguanine-DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.

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The ability to repair damaged DNA was determined in different cell populations of rabbit lung cells isolated by centrifugal elutriation. DNA excision repair, measured as unscheduled DNA synthesis, was examined in in vitro confluent primary cultures. A dose dependent level of DNA excision repair was observed in alveolar type II cells after exposure to the direct acting alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and methyl methansesulphonate. Furthermore, O6-alkylguanine-DNA alkyltransferase activity was easily detectable in alveolar type II cells and alveolar macrophages. In contrast, non-ciliated (Clara) cells had 4 to 20-fold lower levels of DNA excision repair and non-detectable levels of O6-alkylguanine-DNA alkyltransferase. Uracil-DNA glycosylase activities in Clara cells and alveolar type II cells were in the same range and had 3-fold lower activity than alveolar macrophages. Our findings indicate that various lung cells differ in DNA repair capacity and may thus differ in sensitivity to some carcinogens.

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A rapid assay of O6-MeG-DNA methyltransferase activity is described. Following incubation of cell extracts with O6-[3H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive protein by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O6-MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although five tumor tissues had 28-84% lower activity of O6-MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O6-MeG-DNA methyltransferase activity in human tumours appears not to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were also observed.

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Furocoumarins from chloroform extraction of Heracleum laciniatum were separated by high pressure liquid chromatography (HPLC) with microparticulate silica gel column investigating different concentrations of acetonitrile and water, acetonitrile and methanol and acetonitrile, methanol and water for mobile phase. With a mixture of 42:43:15 of the latter for mobile phase optimal separation was obtained of the 6 furocoumarins from the plant. Removal of lipid fractions of plant extractions with hexane is recommended to avoid damage to the HPLC column.

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Extracts of human epidermis prepared by the suction blister method were used to measure O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities. Although both activities were detected in all extracts examined, a 4-5-fold interindividual variation in activity was found. No obvious correlation of the two enzyme activities with the age of the patient was observed. Neither was there any correlation between the level of uracil-DNA glycosylase activity and O6-methylguanine-DNA methyltransferase activity.

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The activities of the DNA repair enzymes O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, and the replicative enzyme DNA polymerase alpha, were measured in extracts of human fetal tissues at 18-20 weeks of gestation. In general, O6-methylguanine-DNA methyltransferase activities in fetal tissues were in the same range as in the corresponding adult tissues, except for fetal liver which had approximately 5-fold lower activity. Uracil-DNA glycosylase was, surprisingly, approximately 4-fold lower in fetal tissues compared with adult tissues. Since a critical factor in carcinogenesis may be the rate of repair relative to DNA replication, the activities of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase were compared with the DNA polymerase alpha activity in the same extract. When expressed in this way, O6-methylguanine-DNA methyltransferase activity was lowest in liver and brain and 2- to 14-fold higher in kidney, lung, colon, stomach, small intestine and pancreas. The ratio of uracil-DNA glycosylase to DNA polymerase alpha varied less between different organs. These findings indicate that several fetal organs may be more sensitive than adult organs to some alkylating agents that are known to occur in the environment. Furthermore, the lower capacity of DNA repair is not restricted to repair of alkylation damage, since the activity of uracil-DNA glycosylase is also lower than in adult tissues.

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As a step towards understanding the significance of DNA repair enzymes in the protection against genotoxic and carcinogenic agents, we have examined the activity of O6-methyl-guanine-DNA methyltransferase and uracil-DNA glycosylase in adult human liver, stomach, small intestine and colon. Liver had on average a 5- to 8-fold higher activity of O6-MeG-DNA methyltransferase than the other organs and showed about an 8-fold inter-individual variation. In colon and small intestine an even larger inter-individual variation was observed (10- and 40-fold, respectively). In two colon tumors examined the activity of O6-MeG-DNA methyltransferase was several fold higher than in non-neoplastic colon mucosa from the same individuals, while uracil-DNA glycosylase activity was essentially equal in neoplastic and non-neoplastic tissues. O6-MeG-DNA methyltransferase activities in two gastric tumors examined were not higher than in average non-neoplastic tissue. In general the activity of uracil-DNA glycosylase did not correlate with the O6-MeG-DNA methyltransferase activity. The inter-individual variation of this enzyme in the activity was only 3-fold in liver and normal stomach, but varied 5.5 and 60-fold in colon and small intestine, respectively. In conclusion, we have found that O6-MeG-DNA methyltransferase as well as uracil-DNA glycosylase activity vary considerably between different tissues as well as between different individuals. Whether this variation has a genetic basis or reflects variation in 'life style' is not known.

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dITP may be generated from dATP by a slow, nonenzymatic hydrolysis. While [3H]dITP was degraded rapidly to [3H]deoxyinosine by HeLa cell nuclear extracts, no net degradation of [3H]dITP was observed in the presence of physiological concentrations of ATP, apparently because the extract contained deoxynucleoside diphosphate kinase activity that regenerated [3H]dITP from [3H]dIDP. Isolated HeLa cell nuclei, as well as partially purified DNA polymerase alpha, incorporated [3H]dITP into DNA at 50-60% of the rate of [3H]dGTP incorporation. No rapid release of the incorporated radioactivity was observed. The molecular weight of nascent DNA containing dIMP residues, however, decreased slightly after prolonged incubation in the presence of EDTA, suggesting that a repair process is initiated in dIMP-containing chromatin. Furthermore, release of free [3H]hypoxanthine from [3H]dIMP-containing DNA was detected after incubation with nuclear extracts in the presence of EDTA, suggesting the presence of hypoxanthine-DNA glycosylase activity in HeLa cell nuclei.

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Extracts from HeLa S3 cells, human liver, and rat liver were found to contain an activity that transfers the methyl group from O6-methyl-guanine residues in DNA to a cysteine residue of an acceptor protein. The molecular weights of the acceptor proteins in HeLA cells and human liver are 24,000 +/- 1,000 and 23,000 +/- 1,000, respectively. Assuming that each acceptor molecule is used only once, the average number of acceptor molecules in HeLa cells was calculated to be about 50,000. The extracts also contained 3-methyl-adenine-DNA glycosylase activity and 7-methyl-guanine-DNA glycosylase activity, although the latter activity was not detected in extracts from human liver in our assay system. Thus, the three major alkylation products resulting from the effect of methylating agents, such as N-methyl-N-nitroso urea, can all be repaired in animal cells. Pretreatment of HeLa cells with N-methyl-N'-nitro-N-nitrosoguanidine (0.1 micrograms/ml) strongly reduced the capacity of HeLa cell extracts to repair O6-methyl-guanine residues, while the activity of three DNA-N-glycosylases was essentially unaltered. This inactivation was not caused by a direct methylation of the enzyme by the carcinogen. The results demonstrate that the mechanism of repair of O6-methyl-guanine residues in DNA is strikingly similar in E coli and animal cells, including humans.

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Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5'-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159--165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.

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A very simple and rather unusual purification scheme for phospholipase C from Bacillus cereus has been worked out. Air is bubbled vigorously through the bacterial culture and the foam collected. Liquified foam is centrifuged, dialyzed and heated at 74 degrees C for 5 min. After centrifugation, affinity chromatography is carried out on lipoprotein-Sepharose. The enzyme is then thermally denatured by exposure to 85 degrees C for 5 min and the precipitated material well washed and then renatured from solution in 4 M guanidinium chloride. The final enzyme preparations are electrophoretically homogeneous and easy to crystallize. The recovery of activity exceeds 80%.

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