[Show abstract][Hide abstract] ABSTRACT: Changes in global 5-methylcytosine amount in Acer platanoides seeds were measured.•Viability of A. platanoides seeds was tested by germination and seedling emergence assays.•Desiccation of A. platanoides seeds causes sine wave-like alterations in m5C amount.•Alterations in m5C amount represent a specific response of orthodox seeds to drying.
Plant Physiology and Biochemistry 10/2014; · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plants respond to environmental changes by modifying gene expression. One of the mechanisms regulating gene expression is methylation of cytosine to 5-methylcytosine (m(5)C) which modulates gene expression by changing chromatin structure. Methylation/demethylation processes affect genes that are controlled upon environmental stresses. Here, on account of the regulatory role of m(5)C, we evaluate the content of m(5)C in DNA from normal and wound-damaged maize leaves. Wounding leads to a transient decrease of the global DNA methylation level ca 20-30% 1 h after the treatment followed by a return to the initial level within the next hours. Similar results were obtained using of radio-labeled nucleotides separated by Thin Layer Chromatography (TLC) or using m(5)C-specific Enzyme-Linked Immunosorbent Assay (ELISA). Wounding induced in maize leaves a two-step oxidative stress, an early one just after wounding and the second two hours later. It coincides with the transient changes of the cytosine methylation level. In the stress-inducible maize calcium-dependent protein kinase ZmCPK11 gene wounding transiently reduced methylation of cytosines 100 and 126 in the first exon.
Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale. 06/2014; 82C:202-208.
[Show abstract][Hide abstract] ABSTRACT: With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down.
PLoS ONE 01/2014; 9(1):e86673. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pedunculate oak (Quercus robur) is an ecologically and economically important forest tree species which produces seeds that are classified as recalcitrant. Thus, cryopreservation of seed meristems is a method for long-term preservation of this germplasm in gene banks. During cryopreservation, many factors, such as desiccation, cryoprotection and cooling/rewarming, can induce stress in the frozen meristems. In this study, in vitro survival and the global DNA methylation level of plumules after cryoprotection, desiccation and cryostorage was evaluated. Results indicated that both desiccation and storage in liquid nitrogen have negligible influence on DNA methylation status of Q. robur plumules. These findings support the cryopreservation of plumules as an appropriate method for conservation of Q. robur germplasm.
Plant Cell Tissue and Organ Culture 01/2014; 117(1). · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.
PLoS ONE 01/2014; 9(11):e113848. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Due to its small size and high activity, it is used as a model for RNA structure-function relationship studies. Here we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells but shows low activity in vitro. To understand it, we analyzed tertiary structure models of substrate:ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and the leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.
[Show abstract][Hide abstract] ABSTRACT: In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.
PLoS ONE 01/2013; 8(1):e54741. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m(5)C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m(5)C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2-3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m(5)C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds.
PLoS ONE 01/2013; 8(8):e70693. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the key questions of biology is the nature and mechanisms of gene function. It has been 60 years since proposing the right-handed model of DNA double helix in 1953. This discovery was honored with Nobel Prize in 1962 and become a breakthrough in knowing and understanding mechanisms of heredity and genetic code. Since that time a great deal of data have been gathered considering functions, structure and DNA application. It became the basis of modern molecular biology, chemical biology and biotechnology. Today we know, that double helix is characterized by its dynamics and plasticity, which depend on its nucleotide sequence. Chromatin structure and DNA mediated charge transport have a crucial role in understanding mechanisms of its damage and repair. Progress in epigenetics allowed to identify new DNA bases, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine. Design of new catalytic nucleic acids and the nanotechnology field of DNA origami reveal its application potential.
[Show abstract][Hide abstract] ABSTRACT: Hammerhead ribozyme is the smallest naturally occurring catalytic RNA. It is a perfect model for structure-function relation studies. Initially, it was identified as an autocatalytic part of viroid and virusoid genomic RNA. It exists within the genomes of many organisms including human, which makes it the most common autocatalytic motif in the nature. After 25 years of intensive research, there are a lot of data considering its structure, conformational dynamics and an influence of tertiary stabilizing motifs on its stability and properties. Structure of the hammerhead ribozyme is a system of elements that influence each other. The knowledge of ribozyme architecture is outstandingly interesting in the context of rules and logic of design, construction and application of such molecules as spatial molecular constructions. Presence of additional structural motifs distinguishes extended hammerhead ribozyme from the minimal one. Hammerhead ribozyme recognizes complementary RNA and catalyses transesterification after the 5'-NUH-3' sequence. Reaction efficiency depends on an arrangement of atoms of the catalytic core presence of metal ions and other intracellular factors. Innovative and potentially better derivatives of the hammerhead ribozyme are objects of extensive research in the field of molecular medicine.
[Show abstract][Hide abstract] ABSTRACT: Convenient and efficient methods of the synthesis of N(6)- and N(4)-substituted derivatives of adenine and cytosine and their 2'-deoxyribosides were developed. The reactions of either unprotected nucleobases (adenine, cytosine) or unprotected 2'-deoxyribosides with aryl or alkyl aldehydes give corresponding Schiff bases that can be reduced to the target title compounds with high overall yields. In the case of aryl aldehydes the imine derivatives are obtained in the presence of methoxides in methanol and reduced with sodium borohydride. The corresponding reactions with alkyl aldehydes require the use of acetic acid and borane dimethyl sulfide complex instead.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the effect of phloretamide (PA), an apple constituent, on the activation of the Nrf2 transcription factor and the expression of its target genes: glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) in normal human THLE-2 hepatocytes and the hepatoma HepG2 cell line. PA did not show significant cytotoxicity towards THLE-2 cells but such an effect was observed in HepG2 cells (IC(50) ∼200μM). The treatment of cells with PA resulted in the translocation of Nrf2 from cytosol to nucleus in both cell lines, but increased the level of its transcript and protein only in THLE-2 cells. In this cell line an increased level of GSTA, GSTP, GSTT, NQO1 mRNA was also observed. Increased expression of GSTs was confirmed by enhancement of their protein levels. The increase in p53 protein content observed in THLE-2 may be associated with its stabilization induced by the enhancement of NQO1 level. PA did not affect Nrf2, GSTs, NQO1 or HO-1 expression in HepG2 cells. These results suggest that PA has rather chemopreventive than chemiotherapeutic potential and acts similarly as apple dihydrochalcones through the induction of detoxification/antioxidative enzymes.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 10/2012; · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. K(i) values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 μM. These compounds cause also a decrease of 5-methylcytosine (m(5)C) level in DNA of mammalian HeLa and HEK293 cells.
European Journal of Medicinal Chemistry 07/2012; 55:243-54. · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNAPhe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNAPhe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86.
[Show abstract][Hide abstract] ABSTRACT: Tobacco smoking creates health problems which apply not only to individuals and the family but also to different ages and social groups, as well as the national economy. Epidemiologic studies conducted at the Institute of Mother and Child indicated that in Poland 25-30% women smoke during pregnancy. Lead exposure from cigarette smoke may have a negative effect on the transplacental flow of micronutrients and have an adverse influence on the growth and development of the fetus, and then on children. The aim of this study was to estimate the effect of smoking cigarettes on plasma and whole blood lead levels in pregnant women. Material and methods: Eighty healthy pregnant women, patients of the Clinical Department of the Obstetrics and Gynecology Institute of Mother and Child and Warsaw Medical University, were divided into two groups: group I - tobacco smokers and group II- tobacco abstainers according to questionnaire declaration and serum cotinine concentration. Current smokers were defined as those who had smoked 5 cigarettes per day for 2 years before conception and continued smoking during pregnancy. The women exposed to environmental tobacco smoke (smoking spouse or other family members, co-workers) were excluded from the non-smoking group. All pregnant volunteers signed a written, informed consent form, approved by the Institute's Ethical Committee. The concentrations of lead in plasma and whole blood were analyzed using inductively coupled plasma mass spectrometry on spectrometer analyzer ICP MS Elan 6100 (Perking Elmer, Germany). Levels of cotinine in serum were determined by Cotinine Direct ELISA test (Calbiotech Inc. Canada). Results: In the group of smoking mothers the mean serum cotinine concentration was 69.1 μg/L, whereas in the group of tobacco abstainers it was present only in trace amount. In group I we observed a significant positive correlation between serum cotinine and the number of cigarettes smoked daily (r=0.74; p<0.001), as well as the period of smoking before conception (r=0.60; p<0.001). The concentrations of lead in the plasma of smoking women were significantly higher than in the group of tobacco abstainers in each trimester of pregnancy (I trimester: 0.22 μg/dL vs 0.12 μg/dL p<0.01; II trimester: 0.19 μg/L vs 0.10 μg/L p<0.001; III trimester 0.28 μg/ dL vs 0.13 μg/dL p<0.0001). Tobacco smoking mothers also had a higher concentration of lead in whole blood as compared to pregnant non-smoking women. These differences were statistically significant and amounted to 2.15 μg/dL vs 1.28 μg/L in the first, 1.99 μg/dL vs 1.19 μg/dL in the second and 2.11 μg/dL vs 1.58 μg/dL in the third trimester of pregnancy. We observed that the level of lead was correlated with cotinine in blood, as well as with the number of cigarettes and the length of time women smoked before conception. Such an effect was observed in every trimester of gestation. A strong correlation between the number of cigarettes/day and lead concentration in plasma (r=0.57; p<0.001) and whole blood (r=0.54; p<0.001) was found in the third trimester of pregnancy. Conclusions: Tobacco smoking during pregnancy increased the concentrations in maternal blood lead. The level of lead in plasma and whole blood correlated with the degree of intensity of cigarette smoking in the pregnant women studied. It may be a result of influencing the mobilization of calcium from the bone with simultaneous release of lead deposited in the bone. Further studies are required to characterize the effect of higher lead level in the blood of mothers on the risk of premature labor, low birth weight of newborns and their inferior development.
Medycyna wieku rozwojowego 01/2012; 16(3):196-204.
From nucleic acids sequences to molecular medicine., Edited by Erdmann VA, Barciszewski J, 01/2012: chapter RNA technologies for mitochondrial genetics.: pages 313-356; Springer-Verlag Berlin Heidelberg.
[Show abstract][Hide abstract] ABSTRACT: Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the ribosome inhibiting protein Mistletoe Lectin I (ML-I) derived from the European mistletoe, Viscum album, in complex with kinetin has been refined at 2.7Å resolution. Suitably large crystals of ML-I were obtained applying the counter diffusion method using the Gel Tube R Crystallization Kit (GT-R) on board the Russian Service Module on the international space station ISS within the GCF mission No. 6, arranged by the Japanese aerospace exploration agency (JAXA). Hexagonal bi-pyramidal crystals were grown during three months under microgravity. Before data collection the crystals were soaked in a saturated solution of kinetin and diffraction data to 2.7Å were collected using synchrotron radiation and cryogenic techniques. The atomic model was refined and revealed a single kinetin molecule in the ribosome inactivation site of ML-I. The complex demonstrates the feasibility of mistletoe to bind plant hormones out of the host regulation system as part of a self protection mechanism.
Biochimica et Biophysica Acta 10/2011; 1824(2):334-8. · 4.66 Impact Factor