Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 µs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

Acute effects of sono-activated photocatalytic titanium dioxide nanoparticles on oral squamous cell carcinoma.

Sonodynamic therapy (SDT) is a new treatment modality using ultrasound to activate certain chemical sensitizers for cancer therapy. In this study, effects of high intensity focused ultrasound (HIFU) combined with photocatalytic titanium dioxide (TiO2) nanoparticles on human oral squamous cell line HSC-2 were investigated. Viability of HSC-2 cells after 0, 0.1, 1, or 3s of HIFU irradiation with 20, 32, 55 and 73Wcm(-2) intensities in the presence or absence of TiO2 was measured immediately after the exposures in vitro. Immediate effects of HIFU (3s, 73Wcm(-2)) combined with TiO2 on solid tumors were also examined by histological study. Cytotoxic effect of HIFU+TiO2in vitro was significantly higher than that of TiO2 or HIFU alone with the tendency to increase for higher HIFU intensity, duration, and TiO2 concentration in the suspension. In vivo results showed significant necrosis and tissue damage in HIFU and HIFU+TiO2 treated samples. However, penetration of TiO2 nanoparticles into the cell cytoplasm was only observed in HIFU+TiO2 treated tissues. In this study, our findings provide a rational basis for the development of an effective HIFU based sonodynamic activation method. This approach offers an attractive non-invasive therapy technique for oral cancer in future.

Comparative study of the conformational lock, dissociative thermal inactivation and stability of euphorbia latex and lentil seedling amine oxidases.

The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10 degrees C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47-70 degrees C, and a "conformational lock" was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.

Conformational lock and dissociative thermal inactivation of lentil seedling amine oxidase.

The kinetics of thermal inactivation of copper-containing amine oxidase from lentil seedlings were studied in a 100 mM potassium phosphate buffer, pH 7, using putrescine as the substrate. The temperature range was between 47-60 degrees C. The thermal inactivation curves were not linear at 52 and 57 degrees C; three linear phases were shown. The first phase gave some information about the number of dimeric forms of the enzyme that were induced by the higher temperatures using the "conformational lock" pertaining theory to oligomeric enzyme. The "conformational lock" caused two additional dimeric forms of the enzyme when the temperature increased to 57 degrees C. The second and third phases were interpreted according to a dissociative thermal inactivation model. These phases showed that lentil amine oxidase was reversibly-dissociated before the irreversible thermal inactivation. Although lentil amine oxidase is not a thermostable enzyme, its dimeric structure can form "conformational lock," conferring a structural tolerance to the enzyme against heat stress.

Effect of chemical chaperones on glucose-induced lysozyme modifications.

Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.

Shock wave induced cytoskeletal and morphological deformations in a human renal carcinoma cell line.

Effects of shock waves on the morphology and cytoskeleton of a human renal carcinoma cell line (ACHN) were investigated in vitro. ACHN monolayer cultured on a cover slide glass was treated with 10 shots of focused underwater shock waves, with 16 MPa peak pressure at the focal area of a piezoceramic shock wave generator. After exposure to the shock wave, based on the severity of morphological deformations of the treated cells, the monolayer was divided into three morphological areas; focal, marginal and intact. Morphological deformations were found to be associated with disorganization of the intracellular cytoskeletal filaments. Deformation of the cytoskeletal proteins in the treated cells were separately studied with respect to the location of the cells within the three morphological areas. Among three major cytoskeletal proteins, actin and tubulin, but not vimentin, were affected by the shock waves. The deformed cells reorganized their cytoskeletal network within 3 h with a pattern similar to the control, indicating the transient characteristic of the shock wave induced cytoskeletal damage in the surviving cells. The remaining cell fragments on the slide glass, which contained short actin filaments, indicated the important role of shear stress in damaging the cytoskeletal fibers by shock waves.

Development of shock wave assisted therapeutic devices and establishment of shock wave therapy.

In order to exploit systems for shock wave therapy, we are working for the development of clinical devices that are based on the concept of shock waves or related phenomena. The paper describes these new therapeutic devices designed for the minimally invasive approach to vascular thromboloysis, selective dissection of tissues, and drug or DNA delivery. To investigate the response of cells to shock loading, a precise method of shock waves generation in space and time has been developed. This method has been studied for application in cardiovascular therapy, cancer treatment, and cranioplasty in close vicinity of the brain. A laser ablation shock wave assisted particle acceleration device has been developed for delivering drug and DNA into soft targets in the human body. The penetration depth of microparticles observed in the experimental targets is believed to be sufficient for pharmacological treatments. In order to achieve an efficient method for rapid revascularization of cerebral thrombosis, a laser induced liquid jet (LILJ) system has been developed. The LILJ has been successfully applied for selective dissection of soft tissue preserving nerve and blood vessels. The system has been further improved by using piezoelectric actuators to drive the liquid jets, as an alternative to pulse laser.

Amine oxidase from lentil seedlings: energetic domains and effect of temperature on activity.

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).