Mahchid Bamdad

Change in intracellular pH in Tetrahymena pyriformis GL in relation to the toxicity level of the lonophorous antibiotic nigericin

Nigericin, a carboxylic poly ether antibiotic, is toxic towards Tetrahymena pyriformis GL [1, 7, 12, 13]. Nigericin transports K(+) across biological membranes along the gradient. This transport is generally equilibrated by an influx of H(+) ions. The toxicity of nigericin (at low 10 mg · l(-1) and high 30 mg · l(-1) levels) was studied by measuring the intracellular pH of Tetrahymena pyriformis GL by means of two labelled molecules: (14)C-DMO and (3)H-inulin. This measurement of pHi requires prior knowledge of the number of cells and their volume. The results reveal a correlation of the toxicity of nigericin, acidification of the intracellular environment and increase in cell volume leading to the return of pHi to control values.

Microbial conversion of lonophorous antibiotic nigericin by the ciliate Tetrahymena pyriformis

The ionophorous antibiotic nigericin is toxic towards the ciliate Tetrahymena pyriformis GL. At the high concentration of 30 mg/l (sublethal dose), it is bioconverted into several products. The yield of one of these (N1) is appreciably improved with pretreated cells, higher concentrations of nigericin (50 or 100 mg/1), and the presence of a larger ciliate biomass. A structural study of N1 by FAB mass spectrometry combined with tandem mass spectrometry and nuclear magnetic resonance shows it to be an epimer, epinigericin, about half as toxic as nigericin. Bioconversion of nigericin by T. pyriformis may be regarded as a detoxication process.

Measurement of imatinib uptake by flow cytometry

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology‐dependant IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single‐cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response.

Epinigericin toxicity towards Tetrahymena pyriformis GL; changes in cell volume and intracellular pH

A study of the toxicity of epinigericin, an antibiotic ionophor, towards the ciliate Tetrahymena pyriformis showed that this molecule stopped cell division, increased cell volume and led to a more basic intracellular pH. The action of epinigericin was probably linked to its function as an ionophor. The ionic selectivity of this molecule is still not known. The raising of the intracellular pH of ciliates by this antibiotic may be linked to its toxic action and its ion-transport mechanism in Tetrahymena. *** DIRECT SUPPORT *** AG903066 00009

Phosphorylation of spleen tyrosine kinase at tyrosine 348 (pSyk348) may be a marker of advanced phase of Chronic Myeloid Leukemia (CML)

We investigated Syk as a potential marker of CML progression. We observed a significant over-expression of Syk mRNA and constitutive phosphorylation of Syk Y348 in blast cells from six AP or BP-CML, but not in 15 CML in chronic phase. We could follow in vivo the recurrence of pSyk(348) throughout blast cell escape, despite observing storage of dasatinib in blast cells. A combination of dasatinib and R406 did not improve therapeutic efficacy in vitro. Our results strongly suggest that Syk activation could be a relevant biomarker of disease progression and dasatinib resistance but is probably not a molecular target.

Toxicity of the herbicide chlorpropham towards the freshwater ciliated protozoan Tetrahymena pyriformis GL

Summary Chlorpropham (CIPC), widely used in agriculture as a herbicide, displays genotoxic and antimitotic properties. Its toxic action was studied on the ubiquitous freshwater ciliate Tetrahymena pyriformis, in short term (six hours) and longer term (24 hours) experiments. The results show that CIPC induces an inhibition of cell division during the first hours of treatment. A slow recovery of cell multiplication was then observed. CIPC treatment in both short and long-term experiments affected the somatic ciliature pattern equally, with irregular arrangement of kineties, giving daughter cells exhibiting an aberrant distribution of nuclear apparatus. Specific toxic action of CIPC was detected on the macronuclear structure, inducing a segregation and concentration of nucleoli. DNA and RNA synthesis was clearly disturbed in the presence of CIPC, DNA synthesis decreasing by about 20% during the four first hours of treatment, and RNA synthesis by about 50% after 24 hours. Hence CIPC toxicity is lasting, still disturbing cells even after several generations.

Toxicity of Two Pairs of Epimeric lonophorous Antibiotics Towards Tetrahymena pyriformis. Structure-Activity Relationship

Summary Structure-activity relationship of four ionophorous antibiotics, nigericin (C 29 -CH{in2OH)and grisorixin (C 29 -CH 3 ) and their respective epimers, epinigericin and epigrisorixin where the methyl is in axial position on C 28 , was investigated. Toxicity studies of these antibiotics (10 mg/l) were carried out with respect to the population time course of. T. pyriformis in the exponential growth phase, and DNA and RNA synthesis. Results showed different durations of cell division inhibition which increased in the following order: epigrisorixin Tetrahymena , between nigericin/grisorixin and between each antibiotic and its epimer, may be due respectively to structural differences at carbons 30 and 28.

Mechanism of nigericin bioconversion by the ciliateTetrahymena pyriformis GL

SummaryNigericin, an ionophorous antibiotic, is converted to epinigericin by the ciliateTetrahymena pyriformis. Bioconversion occurs in the presence of intact cells but not with broken cells (sonicated ciliates). However, broken cells obtained from intact ciliates pretreated with 1 mg/l of nigericin, before sonication, converted nigericin to epinigericin. Non-impairment after adding actinomycin D and cycloheximide to the ciliate culture suggests that the induction mechanism involves no mRNA synthesis, nor any de novo synthesis of protein. Cell fractionation ofTetrahymena demonstrates that the cytosolic compartment is implicated in this bioconversion.