Enantioselective chromatography of alkyl derivatives of 5-ethyl-5-phenyl-2-thiobarbituric acid studied by semiempirical AM1 method

Complexation of alkyl derivatives of 5-ethyl-5-phenyl-2-thiobarbituric acid (2-thiophenobarbital) enantiomers by beta-cyclodextrin was investigated by the AM1 method. The inclusion complexes of beta-cyclodextrin with neutral and anionic forms of these enantiomers have been modeled and energetically optimized. The chiral discrimination of enantiomers was analyzed in terms of differences in the interaction energies. The calculated interaction energies between each enantiomer of the investigated 2-thiobarbiturates and beta-cyclodextrin confirm the ability of beta-cyclodextrin to act as a mobile phase additive in reversed-phase HPLC to separate enantiomers by liquid chromatography and rationalize their order of elution.     

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.     

Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine

Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.     

A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells

It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation. Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids. According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II. Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids.     

Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.     

Directionality of lambda plasmid DNA replication carried out by the heritable replication complex

There are two ‘pathways’ of replication of λ plasmids in Escherichia coli. One pathway requires the assembly of a new replication complex before replication and the second pathway is based on the activity of the replication complex inherited by one of two daughter plasmid copies after a preceding replication round. Such a phenomenon was postulated to occur also in other replicons, including Saccharomyces cerevisiae autonomously replicating sequences. Here we investigated directionality of λ plasmid replication carried out by the heritable and newly assembled replication complexes. Using two-dimensional agarose gel electrophoresis and electron microscopy we demonstrated that in both normal growth conditions and during the relaxed response to amino acid starvation (when only replication carried out by the heritable complex is possible), bidirectionally and undirectionally replicating plasmid molecules occurred in host cells in roughly equal proportions. The results are compatible with the hypothesis that both complexes (heritable and newly assembled) are equivalent.     

There is no evidence for the existence of complex formation between doxorubicin and glutathione

Doxorubicin is co-transported with glutathione by several multidrug resistance proteins (MRPs). In order to check whether weak non-covalent aggregates between doxorubicin and glutathione can be formed, which might be substrates for the transporter, the effect of glutathione on the partition coefficient of doxorubicin was studied. No evidence of an effect of glutathione (at levels up to 20 microM) on the partition coefficient of doxorubicin was found in the pH range of 4.0-7.4. These results indicate that non-covalent doxorubicin-glutathione complexes do not form.