Acetylpyrene-labelled 7-methylguanine nucleotides: unusual fluorescence properties and application to decapping scavenger activity monitoring.

7-Methylguanosine (m(7)G) nucleotides labelled with acetylpyrene (AcPy) were synthesized as fluorescent mRNA 5' end (cap) analogues. The unique fluorescent properties of m(7)G-AcPy conjugates, different from G-AcPy, can be applied to studying various mRNA cap-related processes including the evaluation of putative inhibitors of DcpS enzyme-a therapeutic target in neuromuscular diseases.

Uptake and phytotoxicity of anthracene and benzo[k]fluoranthene applied to the leaves of celery plants (Apium graveolens var. secalinum L.).

The above-ground parts of celery plants were exposed to two polycyclic aromatic hydrocarbons (PAHs): 3-ring anthracene (ANT) and 5-ring benzo[k]fluoranthene (BkF), and the combination of ANT and BkF. After 43 days of exposure (overall dose of 1325µg/plant), celery plants retained only 1.4% of the total dose of ANT and 17.5% of the total dose of BkF. After exposure to a combination of ANT and BkF (1325µg of each compound per plant), the average ANT concentrations were more than twofold higher in/on leaf blades, whereas BkF levels were insignificantly higher. Under natural photoperiod conditions equivalent to a normal day, the combined application of ANT and BkF to the above-ground parts of celery plants slowed down physicochemical transformations of ANT. A similar effect was observed when PAHs were applied to glass surfaces. The combination of both PAHs probably led to stacking interactions, which decreased volatilization, in particular of ANT. Phytotoxicity of ANT and BkF could not be unambiguously established based on the results of this study. In all analyzed treatments, the chlorophyll content of leaf blades remained unchanged. Foliar application of ANT reduced ascorbic acid levels in all analyzed plant parts and increased the total acidity of celery leaves. In all experimental treatments, the total phenolic content of leaves increased up to 15%. Interestingly, ANT and BkF did not produce cumulative effects when applied in combination (when total PAH concentrations per plant were twofold higher).

Spectroscopy studies of interaction hypericin with an anti-cancer therapy drug doxorubicin.

The presented study was designed to estimate the ability of hypericin to interact with the anticancer drug doxorubicin. The hetero-association of hypericin and doxorubicin was investigated with absorption and fluorescence spectroscopy methods in aqueous solution of DMSO in two-component mixtures: doxorubicin-hypericin and three component mixtures: DNA-doxorubicin-hypericin. The data indicate that hypericin forms complexes with doxorubicin and that the association constants are on the order of 300,000 M-1 in a buffer with 30% DMSO content. The absorption spectra of the hypericin - doxorubicin complexes were examined as well. Owing to its ability to interact with flat aromatic compounds, hypericin may potentially be used as an interceptor molecule to detoxification of patients after chemotherapy.

Water dynamics in eggs by means of Nuclear Magnetic Resonance relaxometry.

1H Nuclear Magnetic Resonance relaxometry has been applied to reveal dynamical properties of water molecules embedded into egg yolk and white of three species: turkey, chicken and quail. Two fractions of water molecules, referred to as confined-water and free-water fractions, have been revealed. It has been demonstrated that translation diffusion of the confined-water fraction is three-dimensional. The dynamics of the confined-water has been quantitatively described in terms of diffusion coefficients and rotational correlation times. The parameters have been compared for egg yolk and white for all the species. In addition to these quantities, the number of the confined-water molecules per unit volume has been provided for all cases. The obtained parameters provide insight into the dynamics of water in eggs of different origin and allow to identify similarities and differences between them in connection to the structure of the network formed by the macromolecular fraction of egg yolk and white.

Natural compounds in the human diet and their ability to bind mutagens prevents DNA-mutagen intercalation.

Human diet may contain many mutagenic or carcinogenic aromatic compounds as well as some beneficial physiologically active dietary components, especially plant food phytochemicals, which act as mutagenesis or carcinogenesis inhibitors. This study compared the binding properties of natural compounds in the human diet (caffeine, theophylline, theobromine, and resveratrol) with a water-soluble derivative of chlorophyll to bind to acridine orange, a known mutagen. An analysis was conducted to determine which substances were effective binding agents and may thus be useful in prevention of chemical-induced mutagenesis and carcinogenesis. Data indicated that in order to bind 50% of the mutagen in a complex, less than twice the concentration of chlorophyllin was needed, the resveratrol concentration was 20-fold higher, while a 1000-fold or even 10,000-fold excess of xanthines were required to bind acridine orange.

Determination of lead, cadmium, and persistent organic pollutants in wild and orchard-farm-grown fruit in northeastern Poland.

The concentrations of the heavy metals lead (Pb) and cadmium (Cd) were determined in berries (blackberry, raspberry, bilberry, wild strawberry), and hazelnuts picked from plants in the wild as well as in fruit (blackberry, raspberry, blueberry) and hazelnuts picked from orchard-farmed plants in northeastern Poland. The levels of seven congeners of polychlorinated biphenyls (PCB(7)), gamma isomer of hexachlorocyclohexane (gamma-HCH), and sum of dichlorodiphenyl trichloroethane and its metabolites (SigmaDDT) were also measured in plants and nuts. In addition, the concentrations of Pb, Cd, PCB(7,) gamma-HCH, and SigmaDDT were determined in the surface samples of soil from the sites of fruit picking. The highest acceptable concentrations based upon Polish standards for Pb and Cd were not exceeded in forest fruit. In wild berries, Pb occurred at a level below the detection limit, whereas the concentration of Cd ranged from 6 to 49 microg/kg fresh weight. The levels were Cd 72 microg/kg fresh weight and Pb 290 microg/kg fresh weight in raspberries from orchard plants and exceeded the maximal acceptable limit of 50 microg/kg for Cd and 200 microg/kg for Pb. The level of Pb at 210 microg/kg fresh weight in hazelnuts from orchard plants also exceeded maximal acceptable limits. Individual samples of fruit, regardless of their origin, were found to contain trace amounts of organic pollutants such as 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p'-DDE) and PCB congeners 101 and 118. All soil samples contained from 3.2 to 14.9 mg/kg dry weight concentrations of Pb and most soil samples also contained Cd. Further, individual soil samples were found to contain high levels of SigmaDDT (145 microg/kg), including p,p'-DDT at a concentration of 67 microg/kg. The concentrations of persistent organic pollutants (POP) in wild and orchard-farm-grown fruit in northeastern Poland were generally below threshold permissible limits, and no correlation was found between levels of contaminants in soils and POP concentrations in fruit.

Cadmium ion-chlorophyll interaction - Examination of spectral properties and structure of the cadmium-chlorophyll complex and their relevance to photosynthesis inhibition.

Chlorophyll was shown to spontaneously form a complex with cadmium, which is incorporated at the central position of the chlorophyll molecule porphyrin ring, where it replaces magnesium. The rate of complex formation depended on the ratio of Cd2+ ions to chlorophyll concentration in the solution. In solutions with chlorophyll concentration of C = 1 × 10-5 M and Cd2+ concentrations of C = 1 × 10-5 M, C = 1 × 10-3 M and C = 9 × 10-3 M, Cd-Chl complex formation was completed after 200 h, 50 h and 33 h, respectively. The formation of Cd-Chl complex followed the second order over all substrates reaction order, first order over Cd2+ concentration and first over Chl concentration. The pseudo second order reaction rate constant k, when Cd2+ concentration was equal Chl concentration have been obtained as k = 1.510 ± 0.023 × 10-4 M-1min-1. Quantum chemistry computations showed that Cd-chlorophyll complex existed in two conformations in the methanol solution with cadmium ion placed either below or above the coordination plane. Two times smaller overlap integral of the Chl fluorescence spectrum with the Cd-Chl absorption spectrum IChl,Cd-Chl= 2.4223 × 10-13 cm3/M in comparison with the overlap integral of the Chl fluorescence spectrum with the Chl absorption spectrum IChl,Chl= 4.6210 × 10-13 cm3/M (twice lower probability of energy transfer Chl∗ → Cd-Chl than Chl∗ → Chl) and lower Förster critical distance for resonance energy transfer: RoChl→Cd-Chl= 46.773 Å, RoChl→Chl= 52.086 Å, indicated that in plants intoxicated with cadmium, taken up from the contaminated soil, the energy transfer between Chl and Cd-Chl in antennas will be disturbed, which may be one of the reasons for the inhibition of photosynthesis.

Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

Evaluation of the characteristics of some plant polyphenols as molecules intercepting mitoxantrone.

This paper contains the results of a study on the interactions between mitoxantrone and some bioactive polyphenols. It has been demonstrated that polyphenols can intercept mitoxantrone. Quercetin shows the highest affinity for complexing with mitoxantrone, in contrast to resveratrol, which shows the lowest affinity. The main process underlying the association between cytostatic and polyphenols occurs in the ground state. The values of the constants of the association reactions between the analysed compounds and mitoxantrone are large enough to generate an evident intercepting effect in the three-component system (mitoxantrone-DNA-polyphenol). The affinity of the analysed plant-origin compounds is approximately 1000-fold weaker than the interaction of mitoxantrone with the DNA, which implies that the presence of these compounds in food should not adversely affect oncological therapy but rather could successfully aid oncological treatment by regulating the quantities of the drug in its active form.

The "interceptor" properties of chlorophyllin measured within the three-component system: intercalator-DNA-chlorophyllin.

In aqueous solutions, in the presence of double-stranded DNA, chlorophyllin (CHL) forms complexes with each of the three DNA intercalators: acridine orange (AO), quinacrine mustard (QM), and doxorubicin (DOX). The evidence for these interactions was obtained by measurement changes in the absorption and fluorescence spectra of the mixtures containing DNA and intercalators during titration with CHL. A model of simple competition between DNA and CHL for the intercalator was used to define the measured interactions. The concentrations of the complexes estimated based on this model were consistent with the concentrations obtained by actual measurement of the absorption spectra. The present data provide further support for the role of chlorophyllin as an "interceptor" that may neutralize biological activity of aromatic compounds including mutagens and antitumor drugs.

The mimetics of antiadhesive peptides as the inhibitors of Mycobacterium kansasii phagocytosis.

The alpha-guanidino acids derived of 15 proteinaceous amino acids, omega-guanidino acids with gradually increased hydrocarbon chains, and amidinated dipeptides, were tested as the mimetics of antiadhesive peptides in Mycobacteria phagocytosis inhibition. The crystal structure of omega-guanidino acids used was determined by X-ray structural analysis. It follows from our experiments that the proper distance between guanidine and carboxyl groups of effector molecules is of decisive importance for their inhibitory activity.

Antiadhesive peptides as the inhibitors of Mycobacterium kansasii phagocytosis.

Initial entry of Mycobacteria into the cells depends upon the formation of a molecular complex between Antigen 85 (Ag85), located on the bacterial cell wall, and serum protein-fibronectin (FN) [Nat. Struct. Biol. 7 (2000) 141; Nat. Struct. Biol. 7 (2000) 94]. Therefore, a way of preventing a Mycobacteria invasion could be to inhibit the interaction between fibronectin and leucocyte cellular receptors of the integrin type. We found that some antiadhesive peptides (such as RGDVY and GRGD), derived of fibronectin and human leucocyte antigen DQ (HLA-DQ) sequences, are in fact very potent inhibitors of Mycobacterium kansasii phagocytosis. This observation may open new prospects in the search for tuberculosis therapy.

Analogs of RGDVY and GRGD peptides inhibit Mycobacterium kansasii phagocytosis.

Continuing our research on Mycobacteria kansasii phagocytosis inhibition, we have examined in that context three series of peptides derived from the RGDVY and GRGD sequences. It was found that the levels of the inhibitory activity depend on the amino acid composition as well as on the particular peptide sequence. Distinct inhibitory activity was found in the case of thymopentin (RKDVY), the active fragment of thymopoietin. In this case the Mycobacterium phagocytosis inhibition should be combined with general immunostimulatory activity of RKDVY peptide. Our examination of a series of GRGDV analogs with a successively prolonged oligo-Gly linker inserted into the peptide chain showed that the distance between the Arg and Asp residues required for such an activity should be about 9A.

The immunosuppressive activity of peptide fragments of vaccinia virus C10L protein and a hypothesis on the role of this protein in the viral invasion.

Our previous studies revealed that the 143-148 fragment of interleukin-1 receptor antagonist (IL-1 Ra) molecule with a Val-Thr-Lys-Phe-Tyr-Phe (VTKFYF) sequence inhibits the interleukin-1 (IL-1) interaction with its cellular receptor. The Val-Thr-Arg-Phe-Tyr-Phe (VTRFYF) sequence of the 322-327 fragment of the C-terminal domain of vaccinia virus protein related to the C10L vaccinia gene shows a very high homology to the 143-148 IL-1 Ra fragment, suggesting a similar inhibitory activity. To test this suggestion, we investigated the inhibitory activity of a series of synthetic peptides derived from 316 to 327 fragment of C10L on the interaction of IL-1 with its receptor. We also tested the peptides for their influence on the humoral and cellular immune response. The results indicate that biological activities of the C10L fragments are similar to those obtained for respective fragments of IL-1 Ra. The C-terminal domain of C10L protein can be easily folded into spatial structure similar to the crystallographic one of IL-1 Ra. Based on the crystallographic structure of IL-1 Ra, we constructed a 3-D model of the C10L protein. According to the model, the Val(322)-Asn(328) sequence is localized on the surface of the molecule and, therefore, it may be involved in the interactions with receptors. Our results indicate that the C10L viral protein can play an important role in vaccinia virus evasion of the host immune system. It may consist in the blockade of IL-1 receptors by the C10L protein, a homologue of the IL-1 Ra.

Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues.

Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2,2,7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7)GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G- and m(3)(2,2,7)G-containing caps (K(as) 2 x 10(6) M(-1) to 7 x 10(6) M(-1)) whereas IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K(as) on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.

Interactions of chlorophyllin with acridine orange, quinacrine mustard and doxorubicin analyzed by light absorption and fluorescence spectroscopy.

The present study was designed to estimate the ability of chlorophyllin (CHL) to interact with two acridine mutagens, quinacrine mustard (QM) and acridine orange (AO), and with the antitumor anthracycline doxorubicin (Dox). To this end, aqueous solutions of QM, AO or Dox during titration with CHL were subjected to spectrophotometry and spectrofluorimetry to detect possible interactions between these reagents. The data indicate that CHL forms complexes with AO, QM or Dox in these solutions. The presence of the complexes was manifested by a bathochromic shift of the absorption spectra, as well as by strong quenching of the fluorescence of each of these mutagens in the presence of CHL. CHL, thus, may serve as an interceptor of these mutagenic acridines in different in vivo or in vitro applications. Its ability to interact with Dox may potentially be utilized to detoxify patients overdosed with this or similar drugs.

[The occurrence of polycyclic aromatic hydrocarbons (PAHs) in infusion of natural coffee, coffee substitute and cocoa]. / Wystepowanie wielopierscieniowych weglowodorów aromatycznych (WWA) w naparach kawy naturalnej, zbozowej i kakao.

The content of polycyclic aromatic hydrocarbons (PAHs) in chosen condiments commercially available was investigated. The concentration of these compounds in infusion of natural coffee, coffee ersatz and cocoa was determined by gas chromatography. Fluoranthene, pyrene and benz(a)anthracene were the PAHs, which the most frequently were present in infusions of unfiltered natural coffee. Assuming the extraction of PAHs into infusion of natural coffee of about several percent, obtained results are in good agreement with the amount of those compounds in coffee beans. These compounds were found at similar concentration in infusions of ersatz coffee. The highest concentrations of investigated PAHs were found in cocoa and their amount was 0.82 mg per litre of beverage. The content of these compounds in cocoa was several times higher then the content of those in infusion of unfiltered natural coffee.

[Determination of chlorinated hydrocarbons in coffee beans]. / Zawartosc chlorowanych weglowodorów w ziarnach kawy.

Chlorinated hydrocarbons (gamma-HCH, DDT and their analogous metabolites) were determined in coffee beans. Four sorts of green coffee beans and 18 sorts of burnt coffee beans were used in the research. The method was based on extraction of fat and its destruction with concentrated sulphuric acid. Chlorinated hydrocarbons were extracted with n-hexane, separated and quantitatively determined by gas chromatography. The presence of chlorinated hydrocarbons was detected in green coffee beans and, in smaller quantities, in burnt coffee beans. The concentration of chlorinated hydrocarbons was lower in medium and darkly burnt coffee beans than lightly burnt coffee. The level of DDT and its metabolites in final product decreased after coffee burning at higher temperatures. After brewing the grind coffee beans the remains of chlorinated hydrocarbons were detected in coffee-grounds at concentration to those found in coffee beans. Drinking of natural coffee does not influence an increase of intake the chlorinated hydrocarbons by human beings.

Synthesis and evaluation of fluorescent cap analogues for mRNA labelling.

We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-ß methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.

The hydrolysis of 5'-CAP dinucleotide analogs: catalysis by bi- and terpyridine complexes of Cu²âº and Zn²âº ions.

The kinetics of the hydrolysis of P(1)-(7-methylguanosinyl-5') P(3)-(guanosinyl-5') triphosphate (m(7)GpppG), P(1)-(7-methylguanosinyl-5') P(4)-(guanosinyl-5') tetraphosphate (m(7)GppppG), and diadenosine 5', 5'( ')-P(1),P(3) -triphosphate (ApppA) in the presence of several Cu(2+) or Zn(2+) ions complexed with bi- or terpyridine has been studied at pH 8.0 and 60 °C. Time-dependent product distributions at various metal complex concentrations have been determined by capillary zone electrophoresis and reversed-phase high performance liquid chromatography. The results show that the predominant hydrolytic reaction is the cleavage of 5',5'-oligophosphate bridge, with Cu(2+) complexes being approximately 15-fold more efficient catalysts than Zn(2+) chelates. In addition, the effect of metal ions complexes at pH 7.0 and 8.0 on the imidazole ring opening in m(7)Gua mononucleotides has been studied. The influence of Cu(2+) complexes on imidazole ring cleavage of mononucleotides is modest, whereas Zn(2+) complexes are almost inactive.