Role of pulse shape in cell membrane electropermeabilization

The role of the amplitude, number, and duration of unipolar rectangular electric pulses in cell membrane electropermeabilization in vitro has been the subject of several studies. With respect to unipolar rectangular pulses, an improved efficiency has been reported for several modifications of the pulse shape: separate bipolar pulses, continuous bipolar waveforms, and sine-modulated pulses. In this paper, we present the results of a systematic study of the role of pulse shape in permeabilization, cell death, and molecular uptake. We have first compared the efficiency of 1-ms unipolar pulses with rise- and falltimes ranging from 2 to 100 μs, observing no statistically significant difference. We then compared the efficiency of triangular, sine, and rectangular bipolar pulses, and finally the efficiency of sine-modulated unipolar pulses with different percentages of modulation. We show that the results of these experiments can be explained on the basis of the time during which the pulse amplitude exceeds a certain critical value.     

Biophysical characterization of an indolinone inhibitor in the ATP-binding site of DNA gyrase

Fighting bacterial resistance is a challenging task in the field of medicinal chemistry. DNA gyrase represents a validated antibacterial target and has drawn much interest in recent years. By a structure-based approach we have previously discovered compound 1, an indolinone derivative, possessing inhibitory activity against DNA gyrase. In the present paper, a detailed biophysical characterization of this inhibitor is described. Using mass spectrometry, NMR spectroscopy, and fluorescence experiments we have demonstrated that compound 1 binds reversibly to the ATP-binding site of the 24 kDa N-terminal fragment of DNA gyrase B from Escherichia coli (GyrB24) with low micromolar affinity. Based on these data, a plausible molecular model of compound 1 in the active site of GyrB24 was constructed. The predicted binding mode explains the competitive inhibitory mechanism with respect to ATP and forms a useful basis for further development of potent DNA gyrase inhibitors.     

How to trigger periplasmic release in recombinant Escherichia coli: A comparative analysis

Recombinant protein production in Escherichia coli usually leads to accumulation of the product inside the cells. To capture the product, cells are harvested, resuspended, and lysed. However, in cases where the product is transported to the periplasm, selective disruption of the outer membrane leads to much purer crude extracts compared to complete cell lysis, as only 4–8% of the native E. coli host cell proteins are located in the periplasmic space. A variety of different strategies to enable selective release of the product from the periplasm is available. However, in most of these studies cells are harvested before they are resuspended in permeabilization agent and no differentiation between leakiness and lysis is made. Here, we tested and compared different strategies to trigger leakiness. In contrast to other studies, we performed these experiments during cultivation and quantified both leakiness and lysis. In summary, we recommend incubation with 350 mM TRIS at constant pH for several hours followed by a mild heat treatment up to 38°C to trigger leakiness with only minimal lysis. This study represents a comparative summary of different strategies to trigger E. coli leakiness and describes a solid basis for further experiments in this field.     

Pulsed electric current enhances the phorbol ester induced oxidative burst in human neutrophils.

Oxidative burst (OB) response in human neutrophils, measured with chemiluminescence (CL), has been used to determine whether pulsed electric current (PEC) might induce a functional response in these electrically nonexcitable cells, and also whether it might modify cellular response to tumor-promoting phorbol ester (PMA). Five minutes of PEC treatment caused no significant changes in neutrophil CL levels in HBSS (1.2 mM Ca2+ concentration) as well as in HBSS-EGTA, where the extracellular Ca2+ concentration was reduced to less than 30 nM. The CL level of PMA-activated neutrophils in HBSS was 52% higher than in HBSS-EGTA. In HBSS the CL level, after the combined PMA and PEC treatment, was 53% higher than in PMA-alone-treated neutrophils. Activation of the OB in HBSS-EGTA with PMA and PEC was 13% higher than in solely PMA treated neutrophils. The results suggest that in neutrophil OB response, the PEC effect is closely related with cellular calcium mobilization, since depletion of extracellular Ca2+ decreased the PEC effect.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Prooxidant/Antioxidant Balance in Hypoxia: A Cross-Over Study on Normobaric vs. Hypobaric “Live High-Train Low”

“Live High-Train Low” (LHTL) training can alter oxidative status of athletes. This study compared prooxidant/antioxidant balance responses following two LHTL protocols of the same duration and at the same living altitude of 2250 m in either normobaric (NH) or hypobaric (HH) hypoxia. Twenty-four well-trained triathletes underwent the following two 18-day LHTL protocols in a cross-over and randomized manner: Living altitude (P_IO₂ = 111.9 ± 0.6 vs. 111.6 ± 0.6 mmHg in NH and HH, respectively); training “natural” altitude (~1000–1100 m) and training loads were precisely matched between both LHTL protocols. Plasma levels of oxidative stress [advanced oxidation protein products (AOPP) and nitrotyrosine] and antioxidant markers [ferric-reducing antioxidant power (FRAP), superoxide dismutase (SOD) and catalase], NO metabolism end-products (NOx) and uric acid (UA) were determined before (Pre) and after (Post) the LHTL. Cumulative hypoxic exposure was lower during the NH (229 ± 6 hrs.) compared to the HH (310 ± 4 hrs.; P<0.01) protocol. Following the LHTL, the concentration of AOPP decreased (-27%; P<0.01) and nitrotyrosine increased (+67%; P<0.05) in HH only. FRAP was decreased (-27%; P<0.05) after the NH while was SOD and UA were only increased following the HH (SOD: +54%; P<0.01 and UA: +15%; P<0.01). Catalase activity was increased in the NH only (+20%; P<0.05). These data suggest that 18-days of LHTL performed in either NH or HH differentially affect oxidative status of athletes. Higher oxidative stress levels following the HH LHTL might be explained by the higher overall hypoxic dose and different physiological responses between the NH and HH.     

Direct analysis of carbohydrates in animal plasma by ion chromatography coupled with mass spectrometry and pulsed amperometric detection for use as a non-invasive diagnostic tool

The present paper demonstrates that electrochemical detection (ECD) coupled to ion chromatography and electrospray ionization tandem mass spectrometry (IC-ECD-ESI/MS/MS) can be used to rapidly estimate some indications of the health status of organisms. The lactulose to mannitol ratio (L/M) is used as a non-invasive assay to investigate small intestinal absorption pathways and mucosal integrity. In the present study, an evaluation of the negative effects of nonsteroidal anti-inflammatory drug meloxicam perorally administrated to a group of dogs was carried out by determining the lactulose/mannitol index using the IC-ECD-ESI/MS/MS hyphenated technique. According to the results of the study, meloxicam altered gastrointestinal permeability. Coenzyme Q(10) (CoQ(10)) was tested to determine if it could prevent meloxicam induced gastrointestinal damage and it was found that CoQ(10) could be an effective preventive treatment. Furthermore, plasma glucose concentration level was determined to be an indirect indicator of the oxidative state in the blood. To find out the beneficial effects of a double antioxidant combination (α-lipoic acid (ALA) and CoQ(10)) on the total glucose level in chickens, ALA and CoQ(10) were provided as food additives in factory farm raised chicken. The results of the pilot study indicate that the glucose level in the plasma of chickens group fed with CoQ(10) and ALA was significantly decreased compared to the control group. Ion chromatography (IC) utilizing pulsed amperometric detection (PAD) was compared to ion chromatography coupled with tandem mass spectrometry (MS/MS) as an analytical tool for monitoring the carbohydrate level in biological fluids. In electrochemical detection, the newly developed two-pulse waveform successfully withstands matrix effects in biological samples. Continuous on-line desalting of the high salt concentrations used as the eluent for carbohydrate separation from the anion-exchange column allows coupling of IC and MS techniques. A make-up solution (0.5mM LiCl) was delivered prior to MS detection for efficient ionization of eluted carbohydrates. Method validation showed that both used techniques are practically comparable and some advantages of each are presented.