Kinetin inhibits protein oxidation and glycoxidation in vitro.

We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.

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.

A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA.

Recently, we have reported the presence of kinetin (N6-furfuryladenine) in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We have also found that kinetin has electrochemical properties which can be used for monitoring the level of this modified base in DNA. Here, for the first time, we propose a mechanism for the formation of kinetin in DNA in vivo, based on the analyses of its mass spectra. Since hydroxy radical oxidation at the carbon 5' of the deoxyribose residue yields furfural, we propose that this aldehyde reacts with the amino group of adenine and, after intramolecular rearrangement, kinetin is formed in vivo. Thus kinetin is the first stable secondary DNA damage product known to date with very well defined cytokinin and anti-aging properties, linked to oxidative processes in the cell. These results also indicate that N6-furfuryladenine or kinetin is an important component of a new salvage pathway of hydroxy radicals constituting a 'free radical sink'. In this way, the cells can neutralize the harmful properties of hydroxyl radical reaction products, such as furfural, and respond to oxidative stress by inducing defence mechanisms of maintenance and repair.

Furfural, a precursor of the cytokinin hormone kinetin, and base propenals are formed by hydroxyl radical damage of DNA.

Recently, we have detected kinetin (N6-furfuryladenine), a well known cytokinin plant hormone, in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We had suggested that the furfuryl moiety of kinetin originates from furfural which is one of the primary oxidation products of deoxyribose in DNA. Here we show that the human cell extracts treated with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA) give rise to oxime derivatives of various aldehydes present in the cell. Mass spectrometric analysis of silylated oximes showed several mass signals of different species, one of which was identified as furfural. Furthermore, detailed inspection of the mass spectra of DNA showed the mass signals of 165, 180, 189 and 206 m/z which correspond to cytosine-propenal, thymine-propenal, adenine-propenal and guanine-propenal, respectively. The presence of furfural, along with four base-propenals in the cell extract, as the primary oxidation products of deoxyribose, suggests that degradation of sugar residues in DNA is one of the major routes of cellular damage in addition to the modification of nucleic acid bases.

Evidence for the presence of kinetin in DNA and cell extracts.

In contrast to the current view that kinetin (N6-furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N6-furfuryladenine has electrochemical properties that can be applied for monitoring this modified base by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass-spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N6-furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.

Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture.

DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.

Different conformations of tRNA in the ribosomal P-site and A-site.

Footprinting studies involving radioactively end-labelled tRNA species bound at either the ribosomal P- or A-site have yielded information that the tRNA's conformation is different in the two sites. Appropriate controls showed the relevance of using poly(U)-directed tRNAPhe binding in the P-site and Phe-tRNAPhe in the A-site. Digestion of the tRNA species was effected by RNases T1, T2 and cobra venom RNase. Experiments were performed with tRNAs 32P-labelled at either end to establish positions of primary cuts more confidently. In addition to the common protection of the aminoacyl-stem and anticodon-arm, footprinting experiments revealed striking differences in the accessibility of the T- and D-loops of tRNAs bound in the P- and A-sites. We observed a more open structure for the tRNA in the A-site. These results are consistent with a dynamic structure of tRNA during the translocation step of protein biosynthesis.

Interaction between dimeric methionyl-tRNA synthetase and methionine accepting tRNAs from E. coli.-- Studies by partial ribonuclease digestion.

Using different ribonucleases we have studied the digestion pattern of the two methionine accepting tRNAs, the initiator tRNAfMet and the elongator tRNAmMet from E. coli. The positions and intensities of cleavages are compared to those obtained when the tRNAs are complexed to methionyl-tRNA synthetase. Our results, in comparison with other studies, suggest a general pattern of interaction between tRNAs and their cognate synthetases including the amino acid stem and the anticodon region. Furthermore a lack of involvement of the central region and especially the extra arm seems to be a unique feature of the initiator tRNAMetf.

The site of interaction of aminoacyl-tRNA with elongation factor Tu.

We have used RNases T1, T2 and A to digest two aminoacyl-tRNAs, Escherichia coli Phe-tRNAPhe and E. coli Met- tRNAMetm both in the naked forms and in ternary complexes with E. coli elongation factor Tu (EF-Tu) and GTP. An analysis of the 'footprinting' results has led to an interpretation that has localized the part of the three-dimensional structure of aminoacyl-tRNA covered by the protein in the ternary complex. In terms of the three-dimensional structure of tRNA established for yeast tRNAPhe, EF-Tu covers the aa-end, aa-stem, T-stem, and extra loop on the side of the L-shaped tRNA that exposes the extra loop.

The effect of chemical modification of the CCA end of yeast tRNAPhe on its biological activity on ribosomes.

Yeast tRNAPhe containing 2-thiocytidine (s2C) at position 75 was alkylated specifically at this residue. The biological activities of alkylated and native tRNAPhe were compared in an Escherichia coli protein-synthesizing system in vitro. The alkylated tRNAPhe proved to be active in all steps involved in the elongation phase but the rate of the peptide transfer reaction was somewhat lower when the alkylated tRNAPhe acted as an acceptor of peptidyl residues as compared to native tRNAPhe. These results raise the possibility for attaching spectroscopic or affinity labels at the s2C-75 residue of tRNAPhe without impairing the activity of the tRNA.

Properies of tRNAPhe from yeast carrying a spin label on the 3'-terminal. Interaction with yeast phenylalanyl-tRNA Synthetase and elongation factor Tu from Escherichia coli.

The 2-thioketo function of tRNAPhe-C-s2C-A in which the penultimate cytidine residue is replaced by 20thiocytidine can serve as a site of specific attachment of spin label. By alkylation of tRNAPhe-C-s2C-A with iodoacetamide or its spin label derivatives tRNAPhe-C-(acm)s2C-A or tRNAPheC-(SL)s2C-A are formed. The enzymatic phenylalanylation of these tRNAsPhe revealed that the 2-position of the penultimate cytidine can be modified without impairing this enzymatic reaction but there exists a sterical limitation for the subsituent on this position beyond which the tRNAPhe:phenylalanyl-tRNA synthetase recognition is not possible. Both Phe-tRNAPhe-C-(acm)s2C-A as well as Phe-tRNAPhe-C(SL)s2C-A form ternary complexes with EF-Tu.GTP. The part of the 3'-terminus of tRNAPhe where the additional substituents are attached is therefore not involved in the interaction with this elongation factor. This could be also demonstrated by ESR measurements of spin labelled tRNAsPhe. The correlation times, tauc, for tRNAPhe-C-(SL)s2C-A, Phe-tRNAPhe-C-(SL)s2C-A and Phe-tRNAPhe-C-(SL)s2C-A.EF-Tu:GTP are essentially identical indicating that the structure of the 3'-end of tRNAPhe is not influenced significantly by aminoacylation or ternary complex formation.