Effect of ClAlPcS(2) photodynamic and sonodynamic therapy on HeLa cells.

Photodynamic therapy (PDT) uses photosensitive substance to provoke a cytotoxic reaction causing a cell damage or cell death. The substances, photosensitizers, are usually derivates of porphyrine or phtalocyanine. Photosensitizers must be activated by light in order to produce reactive oxygen species, mainly singlet oxygen. Sonodynamic therapy (SDT) utilizes ultrasound to enhance a cytotoxic effects of compounds called sonosensitizers. In this study we investigated photodynamic and sonodynamic effect of chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) on HeLa cells. DNA damage, cell viability and reactive oxygen species (ROS) production were assessed to find whether the combination of PDT and SDT inflicts HeLa cells more than PDT alone. We found that the combined therapy increases DNA fragmentation, enhances ROS production and decreases cell survival. Our results indicate that ClAlPcS(2) can act as a sonosentitiser and combined with PDT causes more irreversible changes to the cells resulting in cell death than PDT alone.

Antibacterial properties of modified biodegradable PHB non-woven fabric.

The antibacterial properties of poly(hydroxybutyrate) (PHB) non-woven fabric were explored in this study. The PHB was activated by plasma modification and subsequently processed with either immersion into a solution of nanoparticles or direct metallization. The wettability and surface chemistry of the PHB surface was determined. The thickness of the sputtered nanolayer on PHB fabric was characterized. It was found that plasma modification led to a formation of strongly hydrophilic surface, while the subsequent metallization by silver or gold resulted in a significantly increased water contact angle. Further, it was found that antibacterial activity may be controlled by the type of a metal and deposition method used. The immersion of plasma modified fabric into Ag nanoparticle solution led to enhanced antibacterial efficiency of PHB against Escherichia coli (E. coli). Direct silver sputtering on PHB fabric was proved to be a simple method for construction of a surface with strong antibacterial potency against both Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis). We demonstrated the antibacterial activity of PHB fabric modified by plasma activation and consecutive selection of a treatment method for an effective antibacterial surface construction.

Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries.

An optimized procedure for the fast separation of DNA sequencing fragments in short bare fused-silica capillaries filled with highly alkaline solutions of replaceable linear polyacrylamide is presented. High denaturing abilities of the separation media at pH values over 12 are the main reason for their applications in analyses of ssDNA fragments. Moreover, the alkaline solutions of polyacrylamide provide other advantageous properties: three times higher electrophoretic mobility of ssDNA fragments in comparison to those in urea, negligibly low electroosmotic flow in uncoated capillaries, and an adequate stability to a fast alkaline hydrolysis. The separation power of this procedure is enhanced strongly by using monocarboxy poly(ethylene glycol), a terminator for transient isotachophoresis, which eliminates the electromigration dispersion. A high separation efficiency of our system enables to reduce analysis time to several minutes by decreasing the effective lengths of capillaries to 7 cm. A special sample introduction by diffusion is successfully applied. The experimental results demonstrate a potential of the alkaline electrolytes for an implementation in diagnostic sequencing practice.

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains.

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries.

The methodology and instrumentation for fast denaturing electrophoresis in short capillaries was developed and exemplified by detection of short tandem repeat polymorphism in the endothelin 1 gene. The resolution of two nucleotides, which is required for the detection of a dinucleotide repeat polymorphism, was achieved in a capillary of an effective length of 2.5 cm at a temperature of 600C and an electric field strength of 600 V/cm in 42 s. Thus, the use of denaturing electrophoresis in short capillaries with laser-induced fluorescence detection resulted in a reduction of analysis time by a factor of 200 when compared to the conventional slab gel electrophoresis. The developed methodology and instrumentation is advantageous for an implementation in clinical diagnostics and genetic population screening where fast analytical instrumentation amenable to automation is of paramount importance.

Highly alkaline electrolyte for single-stranded DNA separations by electrophoresis in bare silica capillaries.

A new, highly denaturing electrolyte system based on a solution containing 0.01 M NaOH, 0.0015 M Na2B4O5(OH)4 and a replaceable polymer sieving medium was designed for the separation of single-stranded DNA fragments in bare fused-silica capillaries. Extreme denaturing power, together with the optimized composition of the electrolyte, allows for a separation efficiency as high as 2,300,000 height equivalents to a theoretical plate per meter. Sample denaturation in alkaline solutions provides single-stranded DNA fragments without any intra- or intermolecular interactions at room temperature. Their electrophoretic mobilities were found to be twice those of fragments denatured by dimethylformamide or HCl. This can be interpreted in terms of an increased effective charge on the DNA molecules. The surprisingly weak electroosmosis (6 x 10(-10) m2 V-1 s-1) of polymer solutions at pH 12 or higher is considered to be the result of the dissolution of the silica capillary wall. A highly viscous thin layer of dissolved silica probably causes a shift of the slipping plane further away from the wall to the lower value of the zeta potential. Applications of the electrolyte in clinical diagnostics demonstrate its remarkable properties.

Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser-induced fluorescence detection.

The optimum separation conditions of polymerase chain reaction (PCR) products have been found for high-speed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. DNA fragments obtained after PCR amplification of the region covering the (CA)18 microsatellite repeat in nitron 5 of the gene for FcERIbeta, a high affinity glycoprotein receptor for IgE, located on chromosome 11 (11q13), were analyzed with the aim of investigating the repeat polymorphism. The results of polyacrylamide slab gel electrophoresis (PAGE), agarose gel electrophoresis, CE with absorbance detector and CE with LIF are compared. The CE with LIF proved to shorten analysis time by a factor of 100 when compared to slab gel electrophoresis. CE-LIF utilizes a short capillary with an effective length of 6.3 cm and electric field strength from 100 to 550 V/cm. The respective PCR products of sizes from 116 to 210 base pairs (bp) were analyzed in 3 min.

Analysis of heparin-like pharmaceuticals by capillary zone electrophoresis and isotachophoresis.

Synthetic sulfated bis-lactobionic acid amides are important heparin-like pharmaceuticals. The synthesis of these compounds yields molecules differing in the number of sulfate groups, and, during the isolation procedure, the required species may partially decompose or take in some impurities. This article shows that capillary zone electrophoresis may serve well as an expedient method for the analysis of the above-mentioned pharmaceuticals. Complex-forming equilibria between the analytes and bivalent cations present in the background electrolyte bring selectivity necessary for the separation, and the detection at various wavelengths serves as an aid in the characterization of admixtures, decomposition products, and impurities. Capillary isotachophoresis may also be used for the analysis of these species, bringing about the potential of micropreparation of individual compounds and opening the chance for continuous free-flow electrophoresis.

An improvement of restriction analysis of bacteriophage DNA using capillary electrophoresis in agarose solution.

Seven representatives of the serogroup B Staphylococcus aureus bacteriophages, 29, 53, 55, 83A, 85, phi 11 and 80 alpha, were examined by capillary electrophoresis (CE) for genomic homology using DNA restriction analysis. Genomic DNA of individual bacteriophages was cleaved by HindIII restriction endonuclease, and the resulting restriction fragments were separated by standard horizontal agarose slab gel electrophoresis (SGE) as well as by CE in low-melting-point agarose solutions. The number and size of restriction fragments identified by both methods were compared. The high separation power of CE makes it possible to extend the restriction fragment patterns. In most of the restriction patterns, some additional restriction fragments as small as 150 bp, not identified by SGE, were detected. With respect to speed, high separation efficiency, low sample consumption and automation, CE offers a simple procedure for processing of multiple samples cost-effectively in a reasonable time. The comparison of the complemented restriction patterns of the different phage strains and the subsequent identification of their common fragments leads to a deeper understanding of their phylogenetic relationships. The genome homologies expressed for individual phage pairs in terms of coefficient F values ranged from 15 to 69%. These values are in good accordance with the degree of DNA homology of these phages as determined by DNA hybridization studies and thermal denaturation analysis of DNA by other authors. The total size of each phage genome was estimated by adding the sizes of individual restriction fragments.

Capillary zone electrophoresis with indirect photometric detection in the visible range.

Sensitivity and applicability of commonly used indirect photometric detection in the UV region can be adversely affected by the absorption of the UV light by detected solutes and/or by matrix constituents migrating with them. If the visible light is exploited instead of the UV light, both these disadvantages may be for the most part eliminated, and, moreover, the indirect photometric detection will extend its versatility and sensitivity. Prospects as well as main problems of the approach were tested using inorganic ions as analytes. If organic dyes with a molar light absorption coefficient of the order of 10(6) mol-1 L m-1 was selected as the light-absorbing constituents of the background electrolyte, detection sensitivity comparable with the best reported results was reached for anions; for cations, the detection limit was even lower by two orders of magnitude.

Potential response of liquid ion-exchange membrane electrodes with a weak-acid anion as primary ion, and its dependence on pH.

The potential of a liquid ion-exchange membrane electrode with a weak-acid primary anion has been studied in the pH range 1-12. A mathematical model has been designed for describing the dependence of the potential on pH and including changes of the activity of the primary anion A(-) (of a mono- or dibasic acid) in a test solution, extraction of the membrane ion-exchanger into the test solution by protonation in the acidic region, interference by HSO(-)(4) during adjustment of pH with sulphuric acid.