Marita Gavare

DNA-binding studies of AV-153, an antimutagenic and DNA repair-stimulating derivative of 1,4-dihydropiridine

UNLABELLED The ability to intercalate between DNA strands determines the cytotoxic activity of numerous anticancer drugs. Strikingly, intercalating activity was also reported for some compounds considered to be antimutagenic. The aim of this study was to determine the mode of interaction of DNA with the antimutagenic and DNA repair-stimulating dihydropyridine (DHP) AV-153. DNA and AV-153 interactions were studied by means of UV/VIS spectroscopy, fluorimetry and infrared spectroscopy. Compound AV-153 is a 1,4 dihydropyridine with ethoxycarbonyl groups in positions 3 and 5. Computer modeling of AV-153 and DNA interactions suggested an ability of the compound to dock between DNA strands at a single strand break site in the vicinity of two pyrimidines, which was confirmed in the present study. AV-153 evidently interacted with DNA, as addition of DNA to AV-153 solutions resulted in pronounced hyperchromic and bathochromic effects on the spectra. Base modification in a plasmid by peroxynitrite only minimally changed binding affinity of the compound; however, induction of single-strand breaks using Fentons reaction greatly increased binding affinity. The affinity did not change when the ionic strength of the solution was changed from 5 to 150 mM NaCl, although it increased somewhat at 300 mM. Neither was it influenced by temperature changes from 25 to 40°C, however, it decreased when the pH of the solution was changed from 7.4 to 4.7. AV-153 competed with EBr for intercalation sites in DNA: 116 mM of the compound caused a two-fold decrease in fluorescence intensity. FT-IR spectral data analyses indicated formation of complexes between DNA and AV-153. The second derivative spectra analyses indicated interaction of AV-153 with guanine, cytosine and thymine bases, but no interaction with adenine was detected. CONCLUSIONS The antimutagenic substance AV-153 appears to intercalate between the DNA strands at the site of a DNA nick in the vicinity of two pyrimidines.

Hydrogen‐producing Escherichia coli strains overexpressing lactose permease: FT‐IR analysis of the lactose‐induced stress

The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the “lactose killing” phenomenon. Therefore, the recombinant strain was subjected to selection on lactose‐containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid but with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of Fourier transform‐infrared spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress.

Zirconia nanocrystals as submicron level biological label

Inorganic nanocrystals are of increasing interest for their usage in biology and pharmacology research. Our interest was to justify ZrO2 nanocrystal usage as submicron level biological label in bakers yeast Saccharomyces cerevisia culture. For the first time (to our knowledge) images with sub micro up-conversion luminescent particles in biologic media were made. A set of undoped as well as Er and Yb doped ZrO2 samples at different concentrations were prepared by sol-gel method. The up-conversion luminescence for free standing and for nanocrystals with bakers yeast cells was studied and the differences in up-conversion luminescence spectra were analyzed. In vivo toxic effects of ZrO2 nanocrystals were tested by co-cultivation with bakers yeast.

Influence of growth conditions on hydrophobicity of Lactobacillus acidophilus and Bifidobacterium lactis cells and characteristics by FT-IR spectra

In this study we have found, that the values of cell surface hydrophobicity (CSH) of L. acidophilus LA5 and B. lactis Bb12 cells change in response to varied growth conditions – phase of growth, concentration or type of carbon source, presence of oxygen. An evaluation of FT-IR spectra using cluster and quantitative analyses revealed substantial changes of the chemical composition depending on the CSH level of L. acidophilus LA5 and B. lactis Bb12 cells. Decrease of the carbohydrate level was observed in proportion to the increased CSH values alongside with the elevated protein content of more hydrophobic cells of both cultures. The results of present study could help to specify the appropriate physiological state and environment for L. acidophilus LA5 and B. lactis Bb12 to ensure their probiotic properties.

Anhydrobiosis in yeast: FT‐IR spectroscopic studies of yeast grown under conditions of severe oxygen limitation

Anhydrobiosis is a unique state of living organisms when metabolism is temporarily and reversibly delayed in response to the extreme desiccation of cells. The production of dry active preparations of yeast grown under anaerobic conditions is not currently possible because preparations are extremely sensitive to the dehydration procedure, though they could be very helpful in different biotechnological processes, including bioethanol production. To characterize mechanisms responsible for such sensitivity to the dehydration procedure, Fourier transform infrared spectroscopy was used to study the composition of aerobically grown yeast Saccharomyces cerevisiae resistant to dehydration and grown under conditions of severe oxygen limitation and sensitive to dehydration. Results indicated that significantly lower amounts of lipids in cells, grown under conditions of severe oxygen limitation, may be related to the mechanisms of sensitivity. Dehydration of both resistant and sensitive S. cerevisiae cells was accompanied by similar changes in main cellular compounds. Amounts of nucleic acids and proteins decreased slightly, whereas that of lipids and carbohydrates increased. Artificially reduced sensitivity to dehydration in S. cerevisiae cells, grown under conditions of severe oxygen limitation, led to the increase in the lipid concentration. The chemical composition of S. cerevisiae membranes is proposed to dictate the resistance to dehydration in resistant and sensitive cells.

Application of FT-IR Spectroscopy for Fingerprinting of Zymomonas mobilis Respiratory Mutants

Z. mobilis ATCC 29191 and its respiratory knockout mutants, kat-, ndh-, cytB-, and cydB-, were grown under anaerobic and aerobic conditions. FT-IR spectroscopy was used to study the variations of the cell macromolecular composition. Quantitative analysis showed that the concentration ratios—nucleic acids to lipids, for Z. mobilis parent strain, kat-, ndh-, cytB-, and cydB- strains, clearly distinguished Z. mobilis parent strain from its mutant derivatives and corresponded fairly well to the expected degree of biochemical similarity between the strains. Two different FT-IR-spectra hierarchical cluster analysis (HCA) methods were created to differentiate Z. mobilis parent strain and respiratory knockout mutant strains. HCA based on discriminative spectra ranges of carbohydrates, nucleic acids, and lipids allowed to evaluate the influence of growth environment (aeration, growth phase) on the macromolecular composition of cells and differentiate the strains. HCA based on IR spectra of inoculums, in a diagnostic region including the characteristic nucleic acid vibration modes, clearly discriminated the strains under study. Thus it was shown that FT-IR spectroscopy can distinguish various alterations of Z. mobilis respiratory metabolism by HCA of biomass spectra.

Application of FT-IR Spectroscopy for Investigation of Pink Water Remediation by Pine Bark

This study demonstrates the application of FT-IR spectroscopy for investigating the remediation of pink water with the low cost adsorbent pine bark. The removal of 2,4,6-trinitrotoluene (TNT) from pink water by adsorption to pine bark was accompanied by a reduction in intensities of peaks at 1544 and 1347 cm in the spectra of acetonitrile extracts of the pine bark. Hierarchial cluster analysis differentiated samples with high (30-180 mg/L) and low (0-4 mg/L) TNT concentrations, demonstrating the potential of this approach as a quick screening method for the control of the removal of TNT from pink water. The amount of lignin in pine bark was inversely proportional to the size of the pine bark particles, while the concentration of phenolic hydroxyl groups increases with increasing size of pine bark particles. FT-IR spectra showed that as well as TNT, pine bark can also adsorb nitramine explosives such as RDX and HMX.