a: Control untreated cells;b: 0.008 μg/ml; c: 0.012 μg/ml, i.e., the MIC dose; d: 0.04 μg/ml; e: 0.1 μg/ml; f: 0.5 μg/ml. The width of the dispersion of the fragments from the www.selleckchem.com/products/cb-5083.html boundary of the nucleoid was quantified using an image analysis system; this measure is a simple and reliable quantitative parameter that reflects the level of CIP-induced DNA damage (Table 1). Differences were significant between the
doses tested from 0.012 GW-572016 mw μg/ml, except between 0.012 μg/ml and 0.02 μg/ml, between 0.04 μg/ml and 0.08 μg/ml, and between 0.5 μg/ml and 1 μg/ml. Using the images obtained, the nucleoids were categorized into five classes of damage, as shown in Fig. 2 and Table 1: class 0: undamaged, dose of 0 to 0.008 μg/ml (Figs 1a and selleck screening library 1b); class I: low damage level, dose of 0.012 or 0.02 μg/ml (Fig. 1c); class II: intermediate level, dose of 0.04 or 0.08 μg/ml (Fig. 1d); class III: high level, dose of 0.1 μg/ml (Fig. 1e); and class IV: massive fragmentation, doses of 0.5 or 1 μg/ml or higher (Fig. 1f). This latter class of damage was practically undistinguishable from that shown by nucleoids with extensive DNA fragmentation always present spontaneously in cultures . Classification into classes is standard practice in mutagenesis
studies and provides a perceptive description that is especially useful when heterogeneity in the DNA damage rank is evident between the different nucleoids, as observed in the DNA repair experiments. Table 1 Dose-response effect of CIP on TG1 E. coli chromosomal DNA analyzed with the Micro-Halomax® kit. Dose (μg/ml) Width of dispersion (μm) Class Range 0 – 0.003 – 0 0 0.006 – 0.008 – 0.012 1.3 ± 0.3 I ≤ 2.0 0.02 1.6 ± 0.3 0.04 2.5 ± 0.4 II 2.1 – 3.7 0.08 3.3 ± 0.4 0.1 5.1 ± 1.0 III 3.8 – 5.7 0.5 7.8 ± 1.4 IV ≥ 5.8 1 8.8 ± 1.6 The width of the halo of dispersion of DNA fragments is presented in μm (mean ± standard deviation). The extent of DNA damage was classified according to the width of the dispersion.
Meloxicam Figure 2 Nucleoids from E. coli strain TG1 with progressively increased DNA fragmentation after incubation with increasing doses of CIP. 0: undamaged; I: low damage level; II: intermediate damage; III: high damage level; IV: massive fragmentation. Incubation time To determine the minimum incubation time needed to detect a DNA-breakage effect, the TG1 E. coli were collected from LB agar and exposed in liquid LB to 1 μg/ml CIP for 0, 5, 10, 15, 20, 30, and 40 min. The microgel preparation time before immersion in the lysing solution (8 min) must be added to these times because the antibiotic may enter the bacteria and act during this period. Detectable but subtle damage was apparent after 0 min (class I: diffusion width 1.7 ± 0.2 μm) (Fig. 3); this subtle damage appeared as nucleoids with some peripheral DNA fragments unlike in the untreated control cells.