Prospective evaluation of external ocular microbial growth and aqueous humor contamination during cataract surgery.

PURPOSE: To analyze the route of aqueous humor contamination leading to the development of postoperative endophthalmitis. SETTING: Department of Ophthalmology, University of Helsinki, Finland. METHODS: Forty-nine eyes of 49 patients (31 having phacoemulsification and 18 extracapsular cataract extraction [ECCE]) participated in the study. Four bacterial cultures were taken: preoperative conjunctival swab, lid margin culture, intraoperative lacrimal lake sample, and immediate postoperative anterior chamber fluid sample. RESULTS: Preoperative lid margin cultures were positive in 59.2% of eyes, conjunctival cultures in 69.4%, and lacrimal lake cultures in 24.9%. Four aqueous humor samples (8.2%) showed bacterial growth in the anterior chamber aspirate: 3 in the phacoemulsification and 1 in the ECCE group. The bacteria isolated in this study, Staphylococcus epidermidis and Propionibacterium acnes (2 positive isolates each) were sensitive to the preoperative topical antibiotics used. No aqueous humor sample or any from other locations showed gram-negative microbe growth. The most frequently recovered microbes in all samples collected from the 3 other sources were S epidermidis and other coagulase-negative staphylococcus species, followed by P acnes and other propionibacterium species. Staphylococcus aureus, and diptheroids. CONCLUSION: The ocular surface significantly contributed to the transmission of microbes into the eye during cataract surgery. These microbes could not be eradicated by topical preoperative antibiotics. However, no patient developed postoperative endophthalmitis. Natural defense mechanisms appear to fend off a minor inoculum with these microbes of relatively low pathogenicity.

DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture.

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.

DNA methylation in mammalian nuclei.

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.

Effects of DNA binding proteins on DNA methylation in vitro.

The inheritance of DNA methylation patterns may play an important role in the stability of the differentiated state. We have therefore studied the inhibitory effects of DNA binding proteins on DNA methylation in vitro. Mouse L1210 cells grown in the presence of 5-azacytidine acquire hemimethylated sites in their DNA. Purified hemimethylated DNA accepted methyl groups from S-adenosyl-L-methionine in the presence of a crude maintenance methylase more readily than purified DNA isolated from cells not exposed to 5-azacytidine. On the other hand, chromatin fractions isolated from cells grown in the presence or absence of 5-azacytidine were poor substrates for the maintenance methylase irrespective of the number of hemimethylated sites present in the DNA. Inhibition of DNA methylation was shown to be associated primarily with chromatin proteins bound to DNA, and trypsinization of nuclei increased their methyl accepting abilities. Methyl acceptance was increased by salt extraction of chromosomal proteins. These data suggest that association of histones with DNA may play a role in the modulation of methylation patterns.