In addition, the Hep-2 cells were treated with RNAase for 30 min

In addition, the Hep-2 cells were treated with RNAase for 30 min in all periods of infection and incubated with the goat anti-lamin antibodies (diluted 1:800 overnight) washed and exposed for 3 hours to selleck screening library anti-goat immunoglobulin (anti-goat FITC, diluted 1:100). The ureaplasma could be observed close to the nuclear lamin (Figure 2D); however, intranuclear ureaplasmas were not confirmed. The nuclear envelope lamina is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane. This delimitation was important to determine the presence of ureaplasmas in the

perinuclear regions, but not inside the cell nuclei. Gentamicin invasion assay The UB medium promoted the growth of studied ureaplasmas. The exposure of inoculum size of ureaplasmas used for gentamicin allowed no recovery in UB medium. Selleck Dorsomorphin However the ureaplasma of infected Hep-2 cells incubated with gentamicin and trypsinized allowed recovery of this microorganism. In this assay, it was possible to determine that the clinical isolates of ureaplasma revealed to be more concentrated in Hep-2 cells than reference strains. This quantification was determined by 10-fold dilutions of ureaplasma obtained after gentamicin assay in UB medium and expressed as Changing Color Units/ml (CCU/ml). Therefore, the internalization of studied ureaplasma in Hep-2 was confirmed and quantified in this assay. Gentamycin is impermeable to mammalian

cells in the concentration used: it kills only the extra cellular ureaplasma but not the Phosphatidylinositol diacylglycerol-lyase internalized bacteria. The rates of invasion were expressed as buy PLX-4720 the percentage of CCU obtained after

antibiotic exposure relative to the initial inoculum (frequency of invasion). The calculated p-value < 2.2e-16, test for equality of proportions with continuity correction, R project, Vienna, Austria allow for concluding that approximately 1% of the initial inoculum had survived the gentamicin treatment in type-strains and about 10% in clinical isolates. The ATCC strain has a high passage in UB medium. No differences were observed in frequency of invasion between high and low passages clinical isolates (p-value < 2.2e-16). Phospholipase C activity The ureaplasmas were initially cultured at 37°C for 24 hours in one ml of UB broth with pNPPC. The supernatants were evaluated at a wavelength of 405 nm (OD405) in a Multiskan Microplate Reader (Flow Laboratories, Mississauga, Ontario, Canada). The phospholipase C activity was found in the studied ureaplasma and all produced high levels of this enzyme. The average activity was 2,476 to 3,396 pNPPC hydrolysis (U mg-1 protein) (figure 3). This was the highest level that allowed detection of this compound in the present study. The phospholipase C activity also measured in sonicated ureaplasmas cells. The average activity was 0,783 to 0,821 pNPPC hydrolysis (U mg-1 protein). These results showed that most activity is related to secreted enzyme.

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