A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy.

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas' DNA. Modified nucleotides are incorporated into mycoplasmas' DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.

Estradiol dimer inhibits tubulin polymerization and microtubule dynamics.

Microtubule dynamics is one of the major targets for new chemotherapeutic agents. This communication presents the synthesis and biological profiling of steroidal dimers based on estradiol, testosterone and pregnenolone bridged by 2,6-bis(azidomethyl)pyridine between D rings. The biological profiling revealed unique properties of the estradiol dimer including cytotoxic activities on a panel of 11 human cell lines, ability to arrest in the G2/M phase of the cell cycle accompanied with the attenuation of DNA/RNA synthesis. Thorough investigation precluded a genomic mechanism of action and revealed that the estradiol dimer acts at the cytoskeletal level by inhibiting tubulin polymerization. Further studies showed that estradiol dimer, but none of the other structurally related dimeric steroids, inhibited assembly of purified tubulin (IC50, 3.6 µM). The estradiol dimer was more potent than 2-methoxyestradiol, an endogenous metabolite of 17ß-estradiol and well-studied microtubule polymerization inhibitor with antitumor effects that was evaluated in clinical trials. Further, it was equipotent to nocodazole (IC50, 1.5 µM), an antimitotic small molecule of natural origin. Both estradiol dimer and nocodazole completely and reversibly depolymerized microtubules in interphase U2OS cells at 2.5 µM concentration. At lower concentrations (50 nM), estradiol dimer decreased the microtubule dynamics and growth life-time and produced comparable effect to nocodazole on the microtubule dynamicity. In silico modeling predicted that estradiol dimer binds to the colchicine-binding site in the tubulin dimer. Finally, dimerization of the steroids abolished their ability to induce transactivation by estrogen receptor α and androgen receptors. Although other steroids were reported to interact with microtubules, the estradiol dimer represents a new structural type of steroid inhibitor of tubulin polymerization and microtubule dynamics, bearing antimitotic and cytotoxic activity in cancer cell lines.

Lupane and 18α-oleanane derivatives substituted in the position 2, their cytotoxicity and influence on cancer cells.

Lupane derivatives containing an electronegative substituent in the position 2 of the skeleton are often cytotoxic, however, the most active compounds are not selective enough. To further study the influence of a substituent in the position 2 in lupane and 18α-oleanane derivatives on their biological properties, we prepared a set of 38 triterpenoid compounds, 19 of them new, most of them substituted in the position 2. From betulin, we obtained 2-bromo dihydrobetulonic acid and 2-bromo allobetulon and their substitutions yielded derivatives with various substituents in the position 2 such as amines, amides, thiols, and thioethers. Nitration of allobetulon and dihydrobetulonic acid gave 2-nitro and 2,2-dinitro derivatives. Fifteen derivatives had IC50 < 50 µM on a chemosensitive CCRF-CEM (acute lymphoblastic leukemia) cell line and were tested on another seven cancer cell lines including resistant and two non-cancer lines. 2-Amino allobetulin had IC50 4.6 µM and caused significant block of the tumor cells in S and slightly in G2/M transition and caused strong inhibition of DNA and RNA synthesis at 5 × IC50. 2-Amino allobetulin is the most active derivative of 18α-oleanane skeletal type prepared in our research group to date.

Combination of prednisolone and low dosed dexamethasone exhibits greater in vitro antileukemic activity than equiactive dose of prednisolone and overcomes prednisolone drug resistance in acute childhood lymphoblastic leukemia.

INTRODUCTION: Glucocorticoids, particularly prednisone/ prednisolone and dexamethasone, play a prominent role in the treatment of pediatric patients with acute lymphoblastic leukemia due to their ability to induce apoptosis in susceptible cells. Current therapeutic protocols use prednisone for both the prophase and the induction phase of the therapy because the greater antileukemic activity of dexamethasone is compromised by its high frequency of serious adverse reactions. AIM: To compare, for the first time, the in vitro antileukemic activity of prednisolone alone to that of a combination of prednisolone and dexamethasone using dexamethasone at a very low and presumably safe dosage (1/50 w/w). METHODS: Lymphoblasts were isolated from bone marrow and/or blood samples from children with newly diagnosed acute lymphoblastic leukemia. The cytotoxic activity of prednisolone, dexamethasone and the prednisolone/dexamethasone combination against isolated leukemia cells was analyzed using the MTT cytotoxicity assay. RESULTS: We observed differences in the in vitro antileukemic activity of prednisolone and dexamethasone in 21% of the tested patients. 3% of the children were prednisolone sensitive but dexamethasone resistant, while 18% were prednisolone resistant and dexamethasone sensitive. 32% were sensitive to both glucocorticoids and 18% were resistant to both. Cells from patients with good in vivo responses to prednisone monotherapy were more responsive to prednisolone in vitro than were cells from patients with poor prednisone responses (P<0.07). Importantly, we demonstrated that the use of even a minimal dose (1/50 w/w) of dexamethasone with prednisolone dramatically increases the in vitro anti-leukemic activity of prednisolone (P<0.0006). CONCLUSION: The high inter-individual variability of acute lymphoblastic leukemia responses to glucocorticoids suggest that either patients should be selected for prednisone or dexamethasone treatment on the basis of predictive biomarkers or that prednisone should be used directly in combination with a very low and safe dose of dexamethasone to potentiate its antileukemic activity. The latter option is likely to be cheaper and more efficient, and therefore warrants further clinical investigation to assess its efficacy and safety in treating childhood acute lymphoblastic leukemia.