Vera Maximova

Polylactide Stereocomplex-Based Electrospun Materials Possessing Surface with Antibacterial and Hemostatic Properties

Novel fibrous materials of stereocomplex between high-molecular-weight poly(d- or l-)lactide (HMPDLA or HMPLLA) and diblock copolymers consisting of poly(l- or d-)lactide and poly(N,N-dimethylamino-2-ethyl methacrylate) blocks, respectively (PLLA-block-PDMAEMA or PDLA-block-PDMAEMA), were prepared by solution electrospinning. Fibers with mean diameters ranging from 1400 to 1700 nm were obtained. The stereocomplex formation was evidenced by differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analyses. Annealing at 100 degrees C for 8 h resulted in the appearance of crystalline peaks at 2theta values of 12, 21, and 24 degrees for PLA stereocomplex. X-ray photoelectron spectroscopy (XPS) analyses revealed the gradient composition of the fibers with a surface enriched in tertiary amino groups from PDMAEMA blocks. The availability of tertiary amino groups imparts hemostatic and antibacterial properties to the stereocomplex fibrous materials, as indicated by the performed tests on blood cells and on pathogenic microorganisms.

Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers

The fluorescence properties of newly synthesized homodimeric monomethine cyanine dyes in the presence of biopolymers are investigated. They do not fluoresce in TE buffer and bidistilled water but become strongly fluorescent (Q(F)=0.3-0.9) in the region 530-650 nm when bound to dsDNA and ssDNA. The detection limit of dsDNA is about 1.7 ng/ml. Some of dyes studied are able to distinguish between dsDNA and ssDNA, RNA, BSA in solution and gel electrophoresis. The influence of different factors (temperature, pH and viscosity of the medium, presence of histone) on the formation of the dye-biopolymer complexes is investigated. The results of steady-state and dynamic fluorescence measurements concerning the different types of binding between dyes and biopolymers show that the new dyes are applicable in molecular biology as highly sensitive fluorescence labels.

Antibacterial fluoroquinolone antibiotic-containing fibrous materials from poly(L-lactide-co-D,L-lactide) prepared by electrospinning.

Microfibrous materials based on poly(l-lactide-co-d,l-lactide) (coPLA) and coPLA/poly(ethylene glycol) (PEG) containing a fluoroquinolone antibiotic: ciprofloxacin hydrochloride (Cipro), levofloxacin hemihydrate (Levo) or moxifloxacin hydrochloride (Moxi) were obtained by electrospinning. The presence of Moxi led to an increase in the conductivity of the coPLA and coPLA/PEG spinning solutions and to the preparation of membranes composed of fibers aligned with the collector rotation direction. The one-step incorporation of the antibiotics in the fibers was confirmed by infrared spectroscopy and fluorescence microscopy. The antibiotics were dispersed in the coPLA or coPLA/PEG polymer matrix and the XRD spectra revealed the presence of crystalline phase characteristic of PEG and of the respective antibiotic. It was found that the release profiles of the antibiotics did not depend on the antibiotic nature but were dependent on the fiber composition. The presence of PEG in the fibers allowed a more rapid antibiotic release within the first 2h of release. The performed microbiological tests with Staphylococcus aureus revealed that the coPLA/Cipro, coPLA/PEG/Cipro, coPLA/Levo, coPLA/PEG/Levo, coPLA/Moxi and coPLA/PEG/Moxi mats inhibited the bacterial growth. In addition, the presence of an antibiotic in the mats led to a substantial decrease in the adhesion of the pathogenic microorganism and in the case of the coPLA/PEG/antibiotic series - to prevention thereof.

Fluorescence spectral characteristics of novel asymmetric monomethine cyanine dyes in nucleic acid solutions.

Six new asymmetric monomethine cyanine dyes have been synthesized and their fluorescence characteristics in the presence of nucleic acids studied. The new dyes have no fluorescence of their own in water solutions upon excitation at 480 nm but they become strongly fluorescent in the presence of nucleic acids. The fluorescence maxima of the investigated dyes are found at 525–545 nm when bound to dsDNA and around 600 nm upon binding to RNA and ssDNA. Fluorescence quenching studies with increasing concentrations of NaCl indicate that the cyanine dyes have a mixed (intercalating and groove binding) type of interaction with dsDNA.

Heat shock and osmotically dependent steps by DNA uptake after Escherichia coli electroporation

Currently, the major problem in the genetic transformation is to understand how such a large molecule as the plasmid DNA passes through the cell membrane. To solve this problem we used a simplified experimental model with Escherichia coli and the plasmid pBR322: the DNA-bacteria mixture was electroporated in a sucrose solution at 0 degree C and at fixed electrical parameters; the samples were then directly plated into agar. It was found that the electrically-induced bacterial transformation after pulsing is dependent on two factors: heat shock (delta T) and osmotic stress. Our results indicate that two mechanisms contribute to these effects: (i) thermodiffusion of the DNA across the membrane and (ii) osmotic flows of the medium, containing the DNA, into the interior of the cell.

Fluorescence Characteristics of Variously Charged Asymmetric Monomethine Cyanine Dyes in the Presence of Nucleic Acids

Three asymmetric monomethine cyanine dyes bearing one, two, and three positive charges have been synthesized, and their absorption and fluorescence characteristics in the presence of nucleic acids were studied. The maxima of their longest wavelength absorption band lie between 500 and 520 nm. The dyes do not show fluorescence of their own in TE buffer (pH = 7.5), but become strongly fluorescent (QF = 0.2–0.6) on binding to double-stranded DNA. The fluorescence maxima of the investigated dye-dsDNA complexes are in the region of 530–550 nm. The influence of the dye/DNA ratio on both the position and intensity of the fluorescence maxima of the complexes is investigated.

Constitutive expression of a native human interferon-alpha 1 gene in Escherichia coli

1. A plasmid for constitutive expression of the human interferon-alpha 1 (hIFN-alpha 1) gene in Escherichia coli is constructed on the basis of the cloning plasmid pBR322 using a strong synthetic promoter, synthetic ribosome binding site and a native hIFN-alpha 1 gene excised from a chromosomal clone. 2. The yield of recombinant hIFN-alpha 1 from E. coli LE392 cells transformed with the expression plasmid pJP1R9-hIFN-alpha 1 is evaluated to be 2-6 x 10(7) U/l bacterial culture for metabolic shaker and 6-8 x 10(7) U/l for fermentor.

Influence of interferons on the repair of UV-damaged DNA.

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-α and hrIFN-γ) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-α were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-γ was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent

Preparation, properties and complexation ability of polyoxyethylene-bis-anaesthesine

Abstract Poly(ethylene glycol) derivatives of anaesthesine have been prepared and characterized. Complex formation between poly(acrylic acid) or poly(methacrylic acid) and anaesthesine derivatives in dilute aqueous solutions has been studied by potentiometric titration and viscometry. The presence of the hydrophobic anaesthesine groups stabilizes the polycomplexes. The stability is increased with the introduction of hydrophobic end-groups. Anaesthesine derivatives have been found to be of lower toxicity than anaesthesine. The local anaesthetic effect of anaesthesine has been prolonged by its attachment to a polyoxyethylene chain.

Decreased DNA repair capacity of UV-irradiated cells following interferon treatment.

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-α, IFN-βand IFN-γ on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-α had a stronger inhibitory effect than IFN-γ. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.