Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana.

BACKGROUND: Scopoletin and its glucoside scopolin are important secondary metabolites synthesized in plants as a defense mechanism against various environmental stresses. They belong to coumarins, a class of phytochemicals with significant biological activities that is widely used in medical application and cosmetics industry. Although numerous studies showed that a variety of coumarins occurs naturally in several plant species, the details of coumarins biosynthesis and its regulation is not well understood. It was shown previously that coumarins (predominantly scopolin and scopoletin) occur in Arabidopsis thaliana (Arabidopsis) roots, but until now nothing is known about natural variation of their accumulation in this model plant. Therefore, the genetic architecture of coumarins biosynthesis in Arabidopsis has not been studied before. RESULTS: Here, the variation in scopolin and scopoletin content was assessed by comparing seven Arabidopsis accessions. Subsequently, a quantitative trait locus (QTL) mapping was performed with an Advanced Intercross Recombinant Inbred Lines (AI-RILs) mapping population EstC (Est-1 × Col). In order to reveal the genetic basis of both scopolin and scopoletin biosynthesis, two sets of methanol extracts were made from Arabidopsis roots and one set was additionally subjected to enzymatic hydrolysis prior to quantification done by high-performance liquid chromatography (HPLC). We identified one QTL for scopolin and five QTLs for scopoletin accumulation. The identified QTLs explained 13.86% and 37.60% of the observed phenotypic variation in scopolin and scopoletin content, respectively. In silico analysis of genes located in the associated QTL intervals identified a number of possible candidate genes involved in coumarins biosynthesis. CONCLUSIONS: Together, our results demonstrate for the first time that Arabidopsis is an excellent model for studying the genetic and molecular basis of natural variation in coumarins biosynthesis in plants. It additionally provides a basis for fine mapping and cloning of the genes involved in scopolin and scopoletin biosynthesis. Importantly, we have identified new loci for this biosynthetic process.

Caffeine, pentoxifylline and theophylline form stacking complexes with IQ-type heterocyclic aromatic amines.

Methylxanthines (MTX), in particular caffeine (CAF), are known as the most widely consumed alkaloids worldwide. Many accumulated statistical data indicate the protective effect of CAF intake against several types of cancer. One of the possible explanations of this phenomenon is direct non-covalent interaction between CAF and aromatic mutagen/carcinogen molecules through stacking (π-π) complexes formation. Here we demonstrate that CAF and other MTX, pentoxifylline (PTX) and theophylline (TH), form stacking complexes with carcinogenic imidazoquinoline-type (IQ-type) food-borne heterocyclic aromatic amines (HCAs). We estimated neighborhood association constants (K(AC) of the order of magnitude of 10(2)M(-1)) in neutral and acidic environment and enthalpy changes (ΔH values between -15.1 and -39.8kJ/mol) for these interactions using UV-Vis spectroscopy, calculations based on thermodynamical model of mixed aggregation and titration microcalorimetry. Moreover, using Ames test with Salmonella typhimurium TA98 strain and recently developed mutagenicity assay based on bioluminescence of Vibrio harveyi A16 strain, we demonstrated a statistically significant reduction in HCAs mutagenic activity in the presence of MTX.

Superoxide dismutase is upregulated in Staphylococcus aureus following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment.

BACKGROUND: Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all ß-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance. RESULTS: The effectiveness of photodynamic inactivation towards S. aureus and its Sod isogenic mutants deprived of either of the two superoxide dismutase activities, namely SodA or SodM or both of them showed similar results, regardless of the Sod status in TSB medium. On the contrary, in the CL medium (without Mn++ ions) the double SodAM mutant was highly susceptible to photodynamic inactivation. Among 8 clinical isolates of S. aureus analyzed (4 MRSA and 4 MSSA), strains highly resistant and strains highly vulnerable to photodynamic inactivation were noticed. We observed that Sod activity as well as sodA and sodM transcript level increases after protoporphyrin IX-based photodynamic treatment but only in PDI-sensitive strains. CONCLUSIONS: We confirmed that porphyrin-based photokilling efficacy is a strain-dependent phenomenon. We showed that oxidative stress sensitivity caused by the lack of both Sod enzymes can be relieved in the presence of Mn ions and partially in the presence of Fe ions. The fact that Sod activity increase is observed only in PDI-susceptible cells emphasizes that this is probably not a direct factor affecting S. aureus vulnerability to porphyrin-based PDI.

Evaluation of the role of the pharmacological inhibition of Staphylococcus aureus multidrug resistance pumps and the variable levels of the uptake of the sensitizer in the strain-dependent response of Staphylococcus aureus to PPArg(2)-based photodynamic inactivation.

The emergence of antibiotic resistance among pathogenic bacteria has caused an urgent need for the development of alternative therapeutics. One possibility is a combination of nontoxic photosensitizers (PS) and visible light, recognized as photodynamic therapy. Although it is known that Staphylococcus aureus is susceptible to photodynamic inactivation (PDI), the factors that determine the emerging variation among strains in the response to the treatment remain unclear. Some data indicate that cationic photosensitizing dyes such as phenothiaziniums which vary a lot in the chemical structure might target multidrug resistance pumps. In this study, we analyzed whether the uptake and activity of the multidrug resistance pumps might influence the previously observed variations among the clinical strains to protoporphyrin-derived, amphipilic protoporphyrin diarginate-mediated photodynamic treatment (12 J cm(-2) ). Using a new set of four additionally selected methicillin-resistant and methicillin-susceptible clinical as well as ATCC S. aureus strains we confirmed that the bactericidal effect of the PDI is strain-dependent as it ranged from 0 to 5 log(10) -unit reduction in viable counts. However, neither the variable levels of the uptaken PS nor the pharmacological inhibition of NorA efflux pump explained such a phenomenon.

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II.

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.

Impaired mutagenic activities of MPDP(+) (1-methyl-4-phenyl-2,3-dihydropyridinium) and MPP(+) (1-methyl-4-phenylpyridinium) due to their interactions with methylxanthines.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin causing symptoms that resemble those observed in patients suffering from Parkinson's disease. However, in animal or human organisms, MPTP is converted to MPDP(+) (1-methyl-4-phenyl-2,3-dihydropyridinium) and further to MPP(+) (1-methyl-4-phenylpyridinium); the latter compound is the actual neurotoxin. In this report, we demonstrate that MPDP(+) and MPP(+) can form stacking complexes with methylxanthines (caffeine and penthoxifylline), which leads to significant impairment of the biological activity of these toxins (as measured by their mutagenicity).

Formation of stacking complexes between caffeine (1,2,3-trimethylxanthine) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may attenuate biological effects of this neurotoxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin causing symptoms that may resemble those observed in patients suffering from Parkinson's disease. Therefore, MPTP-treated laboratory animals are currently the most favored models to study therapeutic intervention strategies in this disease. It was demonstrated recently that caffeine (1,2,3-trimethylxanthine) intake decreases the risk of Parkinson's disease in various human populations and attenuates MPTP-induced neurological effects in animal models. Since the effects of caffeine on MPTP-treated animals were mimicked by several antagonists of the adenosine A(2A) receptor, it was suggested that caffeine attenuates MPTP toxicity by blocking this receptor. Here, using microcalorimetry and molecular modeling, we demonstrate that caffeine can form stacking (pi-pi) complexes with MPTP. We found that a biological activity of MPTP (induction of mutations in a microbiological mutagenicity assay), which is completely independent on the A(2A) receptor blockade, is significantly reduced by caffeine. Therefore, we suggest that caffeine may attenuate neurotoxicity of MPTP (and possibly other polycyclic aromatic toxins) and reveal its protective effects on the risk of Parkinson's disease not only by blocking the A(2A) receptor but also by sequestering neurotoxin molecules in mixed complexes, especially in stomach.

Methylxanthines (caffeine, pentoxifylline and theophylline) decrease the mutagenic effect of daunomycin, doxorubicin and mitoxantrone.

Previously performed experiments showed that methylxanthines, especially caffeine, may protect cells against cytostatic or cytotoxic effects of several aromatic compounds. One of the proposed mechanisms of this protection is based on stacking interactions between pi electron systems of polycyclic aromatic molecules. In this work, we demonstrate that caffeine and other methylxanthines--pentoxifylline and theophylline--significantly decrease mutagenicity of the anticancer aromatic drugs daunomycin, doxorubicin and mitoxantrone. The spectrophotometric titration of these aromatic compounds by methylxanthines indicated formation of mixed aggregates. The concentrations of free active forms of the drugs decreased when the concentrations of methylxanthines increased in the mixture. Therefore, likely methylxanthines may play a role of scavengers of the free active forms of daunomycin, doxorubicin and mitoxantrone.

Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline.

Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer. However, biological mechanism of this phenomenon remained unknown. Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals. Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine. Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi. Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above. Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.