Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y.

Many factors affect successful virus propagation and plant defence responses. Heat shock protein (Hsp) expression after heat shock plays an ambiguous role in viral infection. On the one hand, Hsp70 participates in plant defence response; on the other hand, Hsp70 could interact with viral proteins and facilitate virus propagation. Here, we studied metabolic adaptations of Nicotiana tabacum L. subjected to heat shock (42 °C, 2 h) before or after inoculating the plants with Potato virus Y (potyvirus). RT-qPCR and ELISA were used for potyvirus quantification. Hsp70 and Hsp90 isoforms were analysed by Western blotting. Salicylic, quinic and chlorogenic acid content was determined by LC-MS. The activity of Hatch-Slack enzymes (as markers of potyviral infection in tobacco) and glycosidases was assayed. Application of heat shock before or after inoculation showed accelerated potyviral propagation in comparison with only inoculated plants. Plants exposed to heat shock and concurrently inoculated showed higher potyviral content, higher amount of Hsp70, together with late decline of quinic acid content and low chlorogenic acid content. Spread of potyviral infection correlated with enhanced salicylic acid content and activities of enzymes of the Hatch-Slack cycle, α- and ß-galactosidase, α-mannosidase, α-glucosidase and ß-N-acetylhexosaminidase. Heat shock proteins accelerate potyviral propagation. The lower weight cytosolic and mitochondrial Hsp70 (~50-75 kDa) persist throughout the viral infection. Also, the plant defense response results in increase of salicylic and chlorogenic acids but decrease of quinic acid content.

Electrophoretic mobility of the capsid protein of the Plum pox virus strain PPV-Rec indicates its partial phosphorylation.

A double-band SDS-PAGE profile was found reproducible for capsid protein (CP) of Plum pox virus (PPV) isolates belonging to the strain PPV-Rec. The double-band was also present in the virus population multiplied in various plants. A single-lesion passage in a hypersensitive host Chenopodium foetidum showed that its presence was not a result of a mixed infection. We found that the two electrophoretic forms of CP shared identical N-terminus. Therefore, they did not originate from an alternative proteolytic processing, but were different in their posttranslational modification. The slower band of CP could be converted to the faster one by the phosphatase treatment. We assumed that CP protein was present in both phosphorylated and dephosphorylated forms in the infected plants.

The activity and isoforms of NADP-malic enzyme in Nicotiana benthamiana plants under biotic stress.

The activity and presence of isoforms of NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) were studied in non-transgenic and transgenic Nicotiana benthamiana plants containing potyviral gene for helper component protease (HC-pro) and in plants infected by Potato virus Y strain NTN (PVY(NTN)). No significant changes in enzyme activities and isoenzyme pattern were observed due to foreign gene introduction and PVY(NTN) infection. However, the activity and isoenzyme composition of NADP-ME measured in extracts from different parts of the plants showed significant differences. Non-denaturating electrophoresis followed by specific detection of NADP-ME activity in polyacrylamide gel detected the presence of only one isoform in roots and younger leaves. Two isoforms of NADP-ME were detected in older leaves and stem (relative molecular mass approximately 248,000 and approximately 280,000) and three isoforms corresponding to tetramer, dimer and monomer were found in flowers. The activity of NADP-ME and the isoenzyme pattern was discussed in relation to its role in plant metabolism within distinct plant parts.

Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes - similarity between human and rat systems.

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.

Affinity chromatography of bull seminal proteins on mannan-Sepharose.

The interaction of bull seminal plasma proteins and sperm with mannan was investigated using an enzyme-linked binding assay (ELBA). A high mannan-binding activity was found in the protein fraction interacting with heparin. Mannan binding to seminal plasma proteins was inhibited by D-mannose and D-fructose, but not by D-mannose-6-phosphate, D-glucose-6-phosphate, ovalbumin and ovomucoid. Mannan inhibited the binding of bovine zona pellucida glycoproteins both to bull sperm and seminal plasma proteins. Yeast mannan immobilized to divinyl sulfone-activated Sepharose was used for the isolation of mannan-binding proteins. The protein components of this fraction were identified on the basis of relative molecular mass determination and N-terminal amino acid sequencing: RNAase dimer, PDC-109 and a protein homologous to BSP-30K (relative molecular mass 14,500). The isolated proteins were characterized by a high zona pellucida binding activity.

D-fructose-binding proteins in bull seminal plasma: isolation and characterization.

The heparin-binding activity of bull seminal plasma proteins was inhibited by D-fructose, D-glucose, inulin and glycogen; D-galactose, dextran and mannan had no effect. While the ejaculated sperm-heparin interaction was not influenced by the presence of saccharides, the heparin-binding activity of epididymal sperm was inhibited by D-fructose. The results of the binding studies were confirmed by affinity chromatography on immobilized heparin followed by elution with monosaccharides. Proteins adsorbed to a heparin-polyacrylamide column and eluted with D-fructose were analyzed by RP HPLC, SDS electrophoresis and by determination of the N-terminal amino-acid sequence. RNAase dimer, PDC-109 and metalloproteinase inhibitor (TIMP-2) were identified.

Partial antigenic characterization of different potato virus Y NTN strain isolates.

Eight isolates of potato virus Y NTN strain (PVY-NTN) of different origin were studied by means of monoclonal antibodies (MAbs) in non-competitive and competitive enzyme-linked immunosorbent assay (ELISA), and by immunoblot analysis of the viral coat protein (CP). As the MAbs reacted with the denatured viral CP, their epitopes must be continuous. The ELISA data demonstrate that the epitopes are topologically different. The epitopes may be located on the N-terminal part of CP as showed its partial amino acid sequencing and the immunoblot analysis.