Sarah W. Kamau

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

EFFECT OF THE MODULATION OF THE MEMBRANE LIPID COMPOSITION ON THE LOCALIZATION AND FUNCTION OF P-GLYCOPROTEIN IN MDR1-MDCK CELLS

SummaryMultidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-β-cyclodextrin (MβCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MβCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MβCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4° C) to higher density fractions in MβCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp function.

Flow cytometry analysis of the effect of allopurinol and the dinitroaniline compound (Chloralin) on the viability and proliferation of Leishmania infantum promastigotes

BackgroundLeishmaniasis is a major parasitic disease in the tropical regions. However, Leishmania infantum has recently emerged as a very important cause of opportunistic infections for individuals positive for human immunodeficiency virus (HIV). However, there is a lack of in vitro tests for assessing the effect of anti-parasitic drugs on the viability and proliferation of Leishmania infantum. The aim of this study is to assess the efficacy of anti-parasitic drugs like allopurinol and Chloralin on the viability and proliferation of L. infantum promastigotes by utilizing two complementary flow cytometric approaches after exposure of the promastigotes to various concentrations of the drugs.ResultsThe density of the cultures in the presence and absence of allopurinol was determined by haemocytometer enumeration. The two flow cytometric approaches used to monitor the drug effect were: (i) a quantitative method to measure cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and (ii) evaluation of cell viability by dual-staining with the membrane-permeable nuclear stain, SBRY-14 and propidium iodide. It was found that concentrations of allopurinol above 50 μg/ml yielded a clear decrease in the proliferation rate of the promastigotes. However, the viability results showed that about 46.8% of the promastigotes incubated in the presence of 800 μg/ml of allopurinol were still alive after 96 hours. In sharp contrast, more than 90% of promastigotes treated with Chloralin 10 μM (2.7 μg/ml) were dead after 48 hours of treatment. These flow cytometric findings suggest that allopurinol has a leishmaniostatic effect while the dinitroaniline compound (Chloralin) has a leishmaniocidal effect against promastigotes.ConclusionsThe flow cytometric data on proliferation and viability were consistent with results obtained from haemocytometer counts and allowed us to develop a model for assessing in vitro the effects of medicaments like allopurinol and chloralin on L. infantum promastigotes on a cellular level.

Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro

ABSTRACT Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.

Flow Cytometric Assessment of Allopurinol Susceptibility in Leishmania infantum Promastigote

BACKGROUND Leishmaniasis is a major tropical and subtropical parasitic disease. Sodium stibogluconate, N-methyl -D-glucamine antimoniate, amphotericin B, pentamidine, and ketoconazole are drugs used to treat this disease. Some of these drugs cause severe adverse side effects and treatment failures are common. Allopurinol, a purine analog, has been used to treat leishmaniasis, alone or combined with the previously mentioned drugs. Low cost, ease of administration (oral), and lack of toxicity make allopurinol a particularly appealing candidate. METHODS The effect of allopurinol on Leishmania infantum (MCAN/ES/89/IPZ229/1/89, zymodeme MON1) wild-type promastigotes (wt-p229), and an altered form of these promastigotes (allo-p229) resulting from long term in vitro exposure to allopurinol, was determined by [(3)H]-thymidine incorporation assays and by diverse flow cytometric approaches. RESULTS Allopurinol arrested the proliferative capacity of wt-p229 promastigotes, reduced the proportion of viable cells, and decreased their total protein content. In contrast, allo-p229 promastigote proliferation was only slightly decelerated and the proportion of viable cells and the protein content were not affected by the allopurinol treatment. CONCLUSIONS The flow cytometry approach allowed us to demonstrate differences in allopurinol susceptibility of the two promastigote forms, expanding the spectrum of flow cytometry applications in studies of parasite resistance.

Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein–Related Activities

No HeadingPurpose.P-glycoprotein (P-gp), a membrane ATPase expelling many structurally unrelated compounds out of cells, is one of the major contributors to multidrug resistance. It is enriched in cold TritonX-100 insoluble membrane domains (i.e., rafts). The purpose of this work was to characterize the ATPase activities of raft preparations from P388 cells overexpressing P-gp (P388/ADR) or devoid of P-gp (P388) and to establish a P-gp–enriched screening system for P-gp–interfering compounds.Methods.Rafts were extracted with cold TritonX-100. The ATPase activity was characterized in 96-well plates using a fluorescence assay.Results.The ATPase activity per mg protein was about five times higher in P388/ADR rafts than in crude membranes. The anti–P-gp antibody C219 inhibited 20% of the activity in P388/ADR rafts but only about 10% of the activity in P388/ADR crude membranes and had no effect on the activity of P388 rafts. The known P-gp–activating compounds verapamil, progesterone, and valinomycin revealed the typical bell-shaped activity/concentration profiles in P388/ADR rafts, indicative for activation at low compound concentrations and inhibition at concentrations >10 to 100 μM. The inhibitory effect was also observed in P388 rafts.Conclusions.Extracted rafts are rich in functional ATPases. Rafts from P-gp–overexpressing cells display P-gp–typical ATPase activity and provide an easy, P-gp–enriched screening system.

Flow cytometric assessment of drug susceptibility in Leishmania infantum promastigotes.

Flow cytometry can be a valuable tool for parasitology in studies of parasite‐drug interaction and cellular toxicity. This unit presents protocols for assessment of Leishmania promastigote proliferation, viability, and cellular protein content. The cytometric approach permits one to detect, differentiate, and quantify cellular changes in these parasites resulting from drug treatment, in this case allopurinol, as well as demonstrate differences in drug susceptibility between promastigote forms. Rapid and relatively simple flow cytometry replaces incorporation of [3H]‐thymidine, hitherto the most common method for determining cell division.