"Action-at-a distance" of a new DNA oxidative damage product 6-furfuryl-adenine (kinetin) on template properties of modified DNA

N(6)-furfuryladenine (kinetin, K) was shown to have cytokinin activity and antiageing effects. It also appears to protect DNA against oxidative damage mediated by the Fenton reaction. Kinetin was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA preparations from human cell culture, as well as in human urine. A proposed mechanism of kinetin synthesis includes furfural, the oxidative damage product of a 2-deoxyribose moiety of DNA, which reacts with an adenine residue to form N(6)-furfuryladenine at DNA level. The identification of kinetin in plant cell extracts, as well as human urine, suggests its excision from DNA by repair mechanisms. Since such a bulky modification as kinetin induces conformational changes of DNA, this could lead to mutations. Therefore, it was interesting to analyze an effect of kinetin on coding properties of DNA. Chemically synthesized oligodeoxynucleotide (20-mer) containing kinetin AAAACTGCCGTCCTGAKGAT was used as a primer. It was elongated in a polymerase chain reaction (PCR) on a template plasmid pEW1 harboring a 210-bp fragment of DNA derived from the 5' end of HIV mRNA. The PCR product of that length containing kinetin in position 17 from the 5' end was isolated and sequenced. Interestingly, DNA polymerase correctly incorporates thymine opposite of kinetin (an adenine derivative) on the complementary strand, but the misincorporations occur in a vicinity of the modified base.     

Occurrence, biosynthesis and properties of kinetin (N6-furfuryladenine)

In this paper we review the data on the structure and properties of N⁶-furfuryladenine (kinetin, K) accumulated during the last forty years. In 1955, kinetin was isolated from DNA as an artifactual rearrangement product of the autoclaving process. Subsequently, its cytokinin activity has been established, demonstrating a wide variety of biological effects, including those on gene expression, inhibition of auxin action, stimulation of calcium flux, the cell cycle, and as an anti-stress and anti-ageing compound. Recently, our views on this very well known plant hormone have changed. There are new data, which show that it occurs in cellular DNA as the product of oxidative, secondary modification and a secondary reaction of DNA. Also new results on the biological function of kinetin have been reported. Various biological effects produced by this hormone in vitro and in vivo have made kinetin even more scientifically interesting and commercially attractive as an ingredient of many beauty cosmetics.     

Cytotoxic effects of kinetin riboside on mouse, human and plant tumour cells

The cytotoxicity of two plant hormone compounds, kinetin and kinetin riboside, was studied on tumour cells, by colony forming assay with increased amount of cytotoxic molecules. The concentration of inhibitor required to reduce cell growth to 50% was determined for these molecules. Kinetin riboside was shown to only act on M4 Beu human and B16 murine melanoma cells at low concentration (1.5 and 0.2 microM). On mice with leukaemia P388, this product has no effect on the tumour growth, and it appears to be toxic at the dose of 25 mg/kg. Kinetin riboside was also shown to have a cytotoxic effect on plant tumour cells (crown-gall).     

Hormonal control of endoreduplication in sugar beet (Beta vulgaris L.) seedlings growing in vitro

The effect on endoreduplication in sugar beet (Beta vulgaris L.) seedlings of five plant hormones in MS medium, ethylene, 24-epibrassinolide (EBR), gibberellic acid (GA(3) ), kinetin and 1-naphthaleneacetic acid (NAA), as well as a combination of kinetin and NAA at two different concentrations, was studied using flow cytometry. Analyses of DNA content in nuclei of the root, hypocotyl and cotyledons of seedlings growing in vitro were performed during their early development, starting from when the root was 0.5-1.0 cm long until expansion of the first pair of leaves. The proportions of nuclei with different DNA contents were established and the mean C-value calculated. The presence of exogenous plant hormones changed endoreduplication intensity, although to different extents, depending on the organ and developmental stage. Ethylene and NAA stimulated the process, while EBR and kinetin suppressed it and GA did not clearly affect it.     

N(6)-Furfuryladenine, kinetin, protects against Fenton reaction-mediated oxidative damage to DNA

N(6)-Furfuryladenine (kinetin) has been shown to have anti-ageing effects on several different systems including plants, human cells in culture, and fruitflies. Since most of the experimental data point toward kinetin acting as an antioxidant both in vitro and in vivo, and since much evidence supporting a causal role of oxidative damage in ageing is accumulating, we tested the antioxidant properties of kinetin directly. Using 8-oxo-2'deoxyguanosine (8-oxo-dG) in calf thymus DNA as a marker for oxidative damage, we demonstrate that kinetin significantly (P < 0.005) protects the DNA against oxidative damage mediated by the Fenton reaction. Kinetin inhibited 8-oxo-dG formation in a dose-dependent manner with a maximum of 50% protection observed at 100 microM kinetin.     

Salinity and hormone interactions in affecting growth,transpiration and ionic relations ofPhaseolus vulgaris

Addition of either abscisic acid (ABA) or kinetin at 10⁻⁶ M to salinized media (20–120mM NaCl) induced remarkable effects on growth ofPhaseolus vulgaris plants. Whereas ABA inhibited the plant growth and the rate of transpiration, kinetin induced stimulation of both parameters. Moreover, ABA increased proline and phosphorus concentrations in the salinized plants whilst kinetin decreased them. ABA induced stimulation of the transport of K, Ca and Cl from root to shoot, accumulation of K, Na and Cl in root cells and inhibits the transport of Na and accumulation of Ca. Kinetin appeared to inhibit the transport and accumulation of Na and Cl, transport of K, and stimulates the accumulation of K and Ca as well as the transport of Ca. The highest influence of both ABA and kinetin was mostly observed when these hormones were used in combination with the highest concentration of NaCl (120 mM) in the medium.     

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.     

Changes in the mitotic cycle in lateral root meristems of Vicia faba following kinetin treatment

Roots of Vicia faba were treated with H³-Thymidine (1 C/ml) for one hour, either before or after a three hour treatment with a 10^–5 M solution of kinetin. The durations of the mitotic cycle and its constituent phases were then derived from the curves recording the rhythmic entry and exit of labeled cells in prophase, both for the kinetin treated roots and the controls. The rate of DNA synthesis was also determined for the control roots and for roots exposed to H³-Thymidine immediately following treatment with kinetin. — Control values for the durations of the mitotic cycle, G1, S, G2 and mitosis were in agreement with most of the results reported in the literature. Kinetin treatment resulted in an increase in the rate of DNA synthesis, but did not affect the number of cells undergoing DNA synthesis, i.e. kinetin, or a similar compound, may be involved in the control of the rate of DNA synthesis in plant root meristems, but it does not appear to be involved in the control of the initiation of the S period of interphase. — The durations of G1 and G2 are shorter and longer respectively in the kinetin treated roots as compared with the control values. Changes in the durations of these phases of interphase, S, cycle time and the rate of cell proliferation have been discussed with respect to time after kinetin treatment and their possible relationship to carbohydrate metabolism.Research supported by an Assistant Professor Research Grant from the University of Missouri — St. Louis.     

Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro

The growth of crown-gall cells cultured in vitro ( Nicotiana tabacum L. cv. White Burley and Parthenocissus tricuspidata cv. Veitchii) is inhibited by alstonine (BG-8), a plant alkaloid, the anti-cancer effect of which has previously been demonstrated on animals and plants. The growth of normal cells is only slightly affected. The inhibitory effect of BG-8 on crown-gall cells is antagonized by indole-3-acetic acid (IAA) added to the culture medium. Kinetin associated with IAA does not prevent this inhibitory effect. BG-8 present in the culture medium containing the two types of hormones seems to modify the later hormonal requirement of Parthenocissus crown-gall tissues.
BG-8 exhibits high binding affinity for crown-gall DNA and, therefore, strongly inhibits its in vitro synthesis. The alkaloid has practically no effect on DNA from healthy cells. The inhibition by BG-8 is dependent on the level of DNA strand separation and on the origin and nature of the tissues (crown-gall DNA is more destabilized than healthy DNA; DNA from habituated tissues is intermediate). IAA and kinetin have opposite effects on the in vitro strand separation of the DNAs from crown-gall cells and, consequently, antagonistic effects on DNA replication (IAA stimulates and kinetin inhibits). It is possible to establish a close relationship between in situ development of crown-gall tissues of the two species studied (in the presence or absence of BG-8 or cell-growth factors), in vitro DNA synthesis and DNA strand separation.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.     

Alternative routes for the genesis of kinetin: A synthetic intramolecular route from 2′-deoxyadenosine to kinetin

We have tested the possible genesis of kinetin from a 2′-deoxyadenylate unit of DNA by a chemical route involving a head-to-tail transfer of deoxyribose from the 9 to the 3 position of the adenine nucleus via a cyclonucleoside, with subsequent elimination of 1′- and 3′-polar groups and 3 → N⁶ intramolecular rearrangement leading to kinetin. We have also determined quantitatively the per cent conversions to 3-furfuryladenine and/or kinetin of the following under autoclaving conditions at 120°, pH 4, 2 atm, and 4 hr: (1) adenine/furfury alcohol; (2) adenine/2-deoxy-d-ribose; (3) 2′-deoxyadenosine; (4) 3-furfuryladenine; (5) 3,5′-(3′-O-diethylphosphoryl-2′-deoxya-denosine)-cyclonucleoside p-toluenesulfonate. The sequence of reactions involving cyclonucleoside formation and rearrangement has been shown to be a chemically feasible route by which kinetin can be formed, although it is not the only way this cytokinin can be generated.     

Studies on the Growth, Flowering, and Production of Female Sterile Flowers as Affected by Different Levels of Foliar Potassium in Solanum sisymbrifolium Lam: II. INTERACTION BETWEEN FOLIAR POTASSIUM AND APPLIED GIBBERELLIC ACID AND 6-FURFURYLAMINOPURINE

The morphogenetic effects including increased internode extensionand flowering with concomitant formation of female sterile flowersinduced by a relatively low content of potassium in S. sisymbrifoliumare enhanced by applying GA₃. In plants with a high K content,the growth regulator simulates all the effects of a low levelof K without influencing the K content. The morphogenetic effects induced by kinetin include a decreasein internode extension and flowering. The similarity betweenthe effects induced by applying kinetin and those resultingfrom a high level of K in the plant is evident from their mutualadditive effect observed. In plants with a low K content kinetininduced decrease in flowering, suppresses the formation of flowersremaining sterile, and there is a concomitant increment in fertileflowers.     

Autoradiographic and microspectrophotometric studies of DNA synthesis in excised tobacco pith tissue

Summary Pith tissue was cultured on modified White’s nutrient medium supplemented, except for controls, with 2 mg/l of IAA and/or 0,5 mg/l of kinetin. For autoradiographs sections were used from tissue grown on medium containing tritiated thymidine. Nuclear DNA contents (Feulgen) were measured by the microspectrophotometric two-wavelengths method. No fading of Feulgen dye in nuclei was found in 11 weeks, in contrast to considerable fading observed in earlier work when a different batch of basic fuchsin had been employed. Counts of radioactive nuclei in autoradiographs agreed well with microspectrophotometric results on the occurrance of DNA synthesis. In control cultures, with or without tritiated thymidine, DNA doubling took place in about 20% of the nuclei during the first two days but in few, if any, thereafter. It was confirmed that kinetin, as well as IAA, increases the frequency of nuclei undergoing DNA synthesis. However, IAA, in contrast with kinetin, still induced considerable DNA doubling after two days. Continued cell reproduction was maintained only in the presence of both substances. This work has been supported in part by research grants toK. Patau from the US Public Health Service (grant No. C-3313) and the American Cancer Society; and by grants toF. Skoog from the American Cancer Society and the Research Committee of the University of Wisconsin Graduate School with funds from the Wisconsin Alumni Research Foundation.     

Plant uptake of radiocaesium: a review of mechanisms, regulation and application

Soil contamination with radiocaesium (Cs) has a long-term radiological impact because it is readily transferred through food chains to human beings. Plant uptake is the major pathway for the migration of radiocaesium from soil to human diet. The plant-related factors that control the uptake of radiocaesium are reviewed. Of these, K supply exerts the greatest influence on Cs uptake from solution. It appears that the uptake of radiocaesium is operated mainly by two transport pathways on plant root cell membranes, namely the K(+) transporter and the K(+) channel pathway. Cationic interactions between K and Cs on isolated K-channels or K transporters are in agreement with studies using intact plants. The K(+) transporter functioning at low external potassium concentration (often <0.3 mM) shows little discrimination against Cs(+), while the K(+) channel is dominant at high external potassium concentration with high discrimination against Cs(+). Caesium has a high mobility within plants. Although radiocaesium is most likely taken up by the K transport systems within the plant, the Cs:K ratio is not uniform within the plant. Difference in internal Cs concentration (when expressed on a dry mass basis) may vary by a factor of 20 between different plant species grown under similar conditions. Phytoremediation may be a possible option to decontaminate radiocaesium-contaminated soils, but its major limitation is that it takes an excessively long time (tens of years) and produces large volumes of waste.     

Liquid chromatography method for plant and mammalian lignans in human urine

Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC-MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.     

A simple and efficient procedure to improve plant regeneration from protoplasts isolated from long-term cell-suspension cultures of indica rice

Summary A simple and efficient method has been developed to improve plant regeneration from protoplasts isolated from >1-yr-old cell-suspension culture of Indica rice (Oryza sativa) cv. Pusa Basmati 1. A two-step regeneration procedure, involving the transfer of calluses to shoot-regeneration medium containing 1% (w/v) agarose prior to culture on a medium containing 0.4% (w/v) agarose, was found to improve plant regeneration. High concentrations of kinetin in the regeneration medium were also found to be beneficial. The two-step regeneration procedure, combined with a high concentration of kinetin (10.0 mgl⁻¹) in the medium, significantly increased plant regeneration. By this method, even though protoplasts were isolated from over 1-yr-old cell-suspension cultures, protoplast-derived plant regeneration frequency reached 16.1% compared with <4% regeneration frequency without such treatment. Use of a similar protocol might improve plant regeneration from other plant species, especially recalcitrant species.     

Identification of kinetin and kinetin riboside in coconut (Cocos nucifera L.) water using a combined approach of liquid chromatography-tandem mass spectrometry, high performance liquid chromatography and capillary electrophoresis

Kinetin (free base and riboside), which was assumed by many scientists to be a synthetic cytokinin plant growth hormone, has been detected for the first time in the endosperm liquid of fresh young coconut fruits ("coconut water"). To facilitate the study, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of kinetin and kinetin riboside in purified coconut water extract sample. Following a solid-phase extraction of cytokinins in coconut water using C18 columns, the samples were further purified by Oasis MCX columns and analyzed by LC-MS/MS for kinetin and kinetin riboside. Detection by mass spectrometry was carried out using selected reaction monitoring (SRM) mode, by identifying the putative kinetin and kinetin riboside based on their characteristic fragments. Based on a signal-to-noise ratio of 3, the limits of detection in SRM mode were 0.02 microM and 0.005 microM for kinetin and kinetin riboside, respectively. Furthermore, optimal conditions for a baseline chromatographic separation of 18 cytokinin standards by high performance liquid chromatography (HPLC) were developed. The HPLC method had been employed for the confirmation and further fractionation of kinetin in coconut water extracts. The confirmation and fractionation of kinetin riboside was carried out using a further modified HPLC program due to the presence of other interfering material(s) in the sample matrix. Finally, fractions of putative kinetin and kinetin riboside collected from HPLC eluate of coconut water sample were further authenticated by independent capillary zone electrophoresis (CZE) experiment.     

The effect of vitamin E and plant extract mixture composed of carvacrol, cinnamaldehyde and capsaicin on oxidative stress induced by high PUFA load in young pigs

The objective of our study was to determine the antioxidative potential of a plant extract (PE) mixture composed of carvacrol, capsicum oleoresin and cinnamaldehyde against high n-3 polyunsaturated fatty acid (PUFA)-induced oxidative stress in young pigs. Thirty-two weaned castrated male crossbred pigs (BW 10.9 kg; n = 32) were randomly assigned to four dietary treatments (n = 8). The negative control diet (Cont) contained 17.2% energy from fat. Oxidative stress was induced in three of the four experimental groups with the inclusion of n-3 PUFA rich linseed oil. Linseed oil substituted wheat starch in the diet to elevate the amount of energy from fat to 34.1%. One of these diets served as a positive control (Oil), one was additionally supplemented with 271.2 mg/kg of PE mixture and one with 90.4 mg/kg α-tocopheryl acetate (Vit E). After 14 days of treatment, blood and urine were collected for the determination of lipid peroxidation and DNA damage. Lipid peroxidation was studied by plasma malondialdehyde (MDA) concentrations, 24 h urinary MDA and F2-isoprostane (iPF2α-VI) excretion, total antioxidant status of plasma and glutathione peroxidase assays. Lymphocyte DNA fragmentation and 24 h urinary 8-hydroxy-2'-deoxyguanosine excretion were measured to determine DNA damage. Consumption of n-3 PUFA rich linseed oil increased the amount of MDA in plasma and urine, and induced DNA damage in lymphocytes, but did not elevate the amount of iPF2α-VI excreted in the urine. The supplementation with PE and with Vit E did not reduce MDA levels in plasma and urine, but it decreased the percentage of DNA damage in lymphocytes (P < 0.001). The PE reduced the urinary iPF2α-VI excretion in comparison to the Cont diet. The results show that PE and Vit E supplemented to pigs in concentrations of 271.2 mg/kg and 90.4 mg/kg, respectively, can effectively protect pig's blood lymphocytes against oxidative DNA damage, thus suggesting their potentially beneficial effects on the immune system under dietary-induced oxidative stress.     

Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones

Stem cuttings of potato plants (Solanum tuberosum L., cv. Miranda) were cultured in vitro on MS medium with sucrose either without or with addition of indole-3-acetic acid (IAA) or kinetin (K) under red light (R) or blue light (B). Plants on medium without hormones under R were thin, long, with very small leaves, and produced no or only a few microtubers (after longer-lasting cultivations). In B, plants remained short, thick, with large, wellde-veloped leaves and produced a significant amount of microtubers. Darkening of both roots and shoots strongly promoted tuber formation; the tubers were formed on the darkened part of the plant. IAA had no pronounced effect on plant development in B except for slight lengthening of the stem, and, in longer cultivations, slightly enhanced tuber formation as well. In R, IAA brought about several significant effects: stem reduction and induction of tuber formation being the most significant. Kinetin in R increased tuber formation slightly. In B, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root (+ stolons)/shoot ratio. Results are discussed with regard to the possible role of auxins and/or cytokinins in mediating the morphogenetic effects of light.