Differential response of antioxidative systems of maize (Zea mays L.) roots cell walls to osmotic and heavy metal stress.

An analysis of peroxidase and ascorbate oxidase activity, phenolic content and antioxidant capacity of isolated maize root cell walls was performed in controls and plants stressed with polyethylene glycol (PEG) or heavy metals, zinc or copper. Peroxidase activity (oxidative and peroxidative) was more pronounced in the ionic than in the covalent cell wall fraction. PEG induced an increase and Zn(2+) a decrease of both ionically bound peroxidase activities. In the covalent fraction, Cu(2+) decreased oxidative and increased peroxidative activity of peroxidase. Isoelectric focusing of ionically bound proteins and activity staining for peroxidase demonstrated increased intensities and appearance of new acidic isoforms, especially in Zn(2+) and PEG treatments. Most pronounced basic isoforms (pI ~ 7.5) in controls, decreased in intensity or completely disappeared in stressed plants. Ascorbate oxidase activity was significantly increased by PEG and decreased by Zn(2+) treatments, and highly correlated with peroxidase activity. Antioxidant capacity and total phenolics content increased in heavy metal-treated and decreased in PEG-treated plants. Analysis of individual phenolic components revealed p-coumaric and ferulic acids, as the most abundant, as well as ferulic acid dimers, trimers and tetramers in the cell walls; their quantity increased under stress conditions. Results presented demonstrate the existence of diverse mechanisms of plant response to different stresses.

Characterisation of phenol oxidase and peroxidase from maize silk.

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.

Synthesis, dopamine D2 receptor binding studies and docking analysis of 5-[3-(4-arylpiperazin-1-yl)propyl]-1H-benzimidazole, 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazole and their analogs.

5-[3-(4-Arylpiperazin-1-yl)propyl]-1H-benzimidazoles and 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazoles were synthesized and their affinity for the D1, D2 and 5-HT1A receptors examined. They expressed a rather high affinity for the D2 dopamine receptor. The main features of ligand-D2 receptor interactions revealed by docking analyses were: salt bridge between piperazine ring protonated N1 and Asp 86, hydrogen bonds of ligand bezimidazole part with Ser 141, Ser 122 and His 189, edge-to-face interactions of arylpiperazine aromatic ring with Phe 178, Tyr 216 and Trp 182 and hydrogen bond between ethereal oxygen in ethylenoxy ligands and hydrogen of Phe 185 or Trp 115. The most active 5-[2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethoxy]-1,3-dihydro-2H-benzimidazole-2-thione (27) has a maximal number of attractive interactions. A satisfactory correlation between docking of the compounds into the D2 receptor and competition binding results was observed.

Electrostatic surface potential calculation on several new halogenated benzimidazole-like dopaminergic ligands.

We examined the effects of the electron density distribution (electrostatic surface potential; ESP) of several new benzimidazole-type ligands on their binding affinity for the D(1) and D(2) dopamine receptors (DAR). Receptors were prepared from synaptosomal membranes of bovine caudate nuclei. [(3)H]SCH 23390 and [(3)H]spiperone were used as specific radiolabels for the D(1) and D(2) receptors, respectively. The ESP of these compounds was calculated using Gaussian 98 W software. Calculations performed with known dopaminergic ligands showed that the electron density charge in the aromatic ring of these compounds favors a higher binding affinity for the D(2) DAR. This was confirmed by the synthesis of halogenated analogues of several known dopaminergic ligands. Halogenation resulted in an increase in the positive charge of the aromatic part of the molecule. None of the newly synthesized compounds was efficient in displacing [(3)H]SCH 23390 from the D1 DAR. The introduction of chlorine into the molecule led to a higher binding affinity for the D(2) DAR of the new ligands in comparison to both parent compounds and brominated ligands. This difference probably originates from the difference in the sizes of chlorine and bromine atoms, which could influence the interaction of a ligand with the receptor binding site. However, among the new ligands with bromine as a substituent, two compounds (8b and 10b) expressed a higher binding affinity and two of them (9b and 11b) a lower binding affinity for the D(2) DAR, when compared to unsubstituted parent compounds. These results indicate that the electrostatic surface potential of a ligand is an important factor in its interaction with the D(2) DAR and that this should be taken into account during design and synthesis of dopaminergic compounds.

D2 dopaminergic and 5-HT1A serotonergic activity of 2-(1-naphthyl)ethyl- and 2-(2-naphthyl)ethyl amines.

Several tertiary 2-phenylethyl, 2-(1-naphthyl)ethyl and 2-(2-naphthyl)ethyl amines were synthesized and their binding affinities for dopamine D(1), D(2) and serotonin 5-HT(1A) receptors evaluated in radioligand binding assays. All compounds were inactive in D(1) dopamine radioligand binding assay. The 2-(1-naphthyl)ethyl analogues expressed a low but significant binding affinity for the D(2) and moderate one for the 5-HT(1A) receptor subtypes. Most of the remaining compounds expressed binding affinity at the 5-HT(1A) receptor subtype but were inactive in D(2) receptor binding assay. Based on these results and considering the chemical characteristics of the compounds synthesized and evaluated for dopaminergic and serotonergic activity throughout the present study it can be concluded that hydrophobic type of interaction (stacking or edge-to-face) plays a significant role in the formation of receptor-ligand complexes of 2-(1-naphthyl)ethyl amines. This structural motive can be applied to design and synthesize new, more potent dopaminergic/serotonergic ligands by slight chemical modifications.

Superoxide synthase and dismutase activity of plasma membranes from maize roots.

Superoxide synthase and superoxide dismutase activity have been monitored in isolated maize ( Zea mays) root plasma membranes spectrophotometrically by determination of nitro-blue tetrazolium and cytochrome c reduction, respectively. Superoxide production was induced by NADH and NADPH, with similar kinetics and approaching saturation at 0.06 mM in the case of NADPH and 0.1 mM in the case of NADH, with rates of 18.6 +/- 5.0 and 21.8 +/- 7.2 nmol/min. mg of protein, respectively. These activities exhibited a broad pH optimum between pH 6.5 and 7.5. Diphenylene iodonium inhibited about 25% (10 microM DPI) and 40% (100 microM DPI) of this activity, imidazole inhibited about 20%, while KCN, a peroxidase inhibitor, did not show any significant inhibition. Superoxide-dismutating activity was shown to occur in the same isolates and depended on the quantity of plasma membrane protein present. Growth of plants on salicylic acid prior to membrane isolation induced a rise in the activity of both of the enzymes by 20-35%, suggesting their coordinated action.

Some genetic and biochemical characteristics of a new source of maize opaque-2 mutant.

The opaque kernels separated from the F1 of crosses of our opaque-2 strains with normal inbred lines contained 45, 50 and 74 percent more lysine in the whole kernel than the translucent kernels from the same ear. The opaque kernels from these crosses contained almost the same level of lysine as the parental opaque-2 strains.Some of our opaque-2 strains contain 63 to 122 percent more lysine than the tested dent or flint normal inbred lines. In comparison with opaque-2 Purdue, four opaque-2 strains had almost the same lysine content and a strain labelled as SP-1 No. 15 contained 25 percent more lysine and 73 percent more tryptophan. Some of our opaque-2 strains contained 55 to 100 percent more tryptophan in the whole kernel than opaque-2 Purdue.In contrast to lysine content, the transmission of the tryptophan content from opaque-2 strains to opaque kernels from their crosses shows variability.We found weak positive correlation (r = +0,3057) between lysine and tryptophan content in opaque-2 kernels.Our opaque-2 strains had a higher lysine content in the endosperm than did opaque-2 Purdue 22.1 - 36.6% more, and compared with normal lines they had 109.5 - 142.6% more.It is apparent that a new source of opaque-2 mutant gene, which has the same genetic and biochemical characteristics as the opaque-2 mutant discovered by Mertz, Bates and Nelson (1964), has been found in a completely new, genetically divergent, strain derived from a large number of populations.