The application of ab initio molecular orbital theory to structural moieties of carbohydrates

Ab initio molecular orbital calculations were made on methoxymethanol, a model for the hemiacetal and acetal moieties in aldopyranoses and methyl aldopyranosides, thereby improving on the previous calculations using methanediol. The new calculations confirmed the favored conformations already deduced, and gave, for the conformational-energy differences and CO bond-length variations, values more appropriate to the carbohydrate systems, as confirmed by a re-examination of the experimental data from crystal-structure determinations. From the results, it was predicted that the OCH₃ bond in methyl aldopyranosides is lengthened; this is supported by the experimental data. An examination of the angles and bond-lengths in the pyranoid ring and of the linkage bonds of oligosaccharides indicated that similar electronic effects involving the oxygen lone-pair electrons apply to oligo- and poly-saccharides.     

In vitro characteristics of 1-phenyl-3-methyl-4-acylpyrazol-5-ones iron chelators

Iron chelators represent a group of structurally different compounds sharing the ability of iron binding. The group has been evolving in recent years mainly due to novel experimental indications associated with variable requirements for iron chelators. A group of synthetic 1-phenyl-3-methyl-4-acyl-pyrazol-5-ones has been known for many years but data on their potential biological activity are rather limited.In this study, we analysed a series of these compounds for their iron-chelating properties as well as for their effects on iron based Fenton chemistry. For the former ferrozine spectrophotometric method and for the latter HPLC method with salicylic acid were used.All of the tested compounds were very efficient ferric chelators but their ferrous-chelating effects differed according to the acyl substitution. Notwithstanding various ferrous chelation activities, the individual Fe²⁺-affinities were not significantly different through pathophysiologically relevant pH conditions and some of the tested substances were more potent ferrous chelators at pH 4.5 than clinically used standard deferoxamine. Of particular interest is H₂QpyQ /2,6-bis[4(1-phenyl-3-methylpyrazol-5-one)carbonyl]pyridine/ which iron-chelating affinity increased when pH was decreasing. In spite of ferrous chelation differences, most of the tested acylpyrazolones were similarly active powerful inhibitors of Fenton chemistry as deferoxamine.Conclusively, acylpyrazolones are efficient iron chelators and H₂QpyQ may represent a prototype of novel specific chelators designated particularly for chelation at acidic conditions.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

The combined luminol/isoluminol chemiluminescence method for differentiating between extracellular and intracellular oxidant production by neutrophils

To address the question why isoluminol, but not luminol, failed to detect oxidants produced intracellularly, differences between these luminophores were investigated with respect to physicochemical parameters and the character of chemiluminescence signal. Our results showed the isoluminol molecule to be more polar, more hydrophilic and possessing lower ability to form intramolecular bonds than the luminol molecule. Therefore, isoluminol: (i) only slightly pervaded biological membranes; (ii) depended essentially on extracellular peroxidase; (iii) did not produce chemiluminescence in the presence of extracellular scavengers; and (iv) it could be considered a specific detector of extracellular radicals. On the other hand, the physicochemical parameters of luminol and partial resistance of its chemiluminescence to the effect of extracellular inhibitors proved the lipo/hydrophilic character of this luminophore and thus its ability to interact with radicals both outside and inside of cells. The luminol chemiluminescence measured in the presence of extracellular scavengers and the isoluminol chemiluminescence were used with the intention to differentiate the effects of two antihistamine drugs on intra- and extracellular radical formation. In activated human neutrophils, brompheniramine inhibited the extracellular and potentiated the intracellular part of chemiluminescence signal, whereas a reducing effect of loratadine was observed in both compartments.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

Viability, ultrastructure and cytokinin metabolism of free and immobilized tobacco chloroplasts

Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix.     

Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils

Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage. We studied the effect of carvedilol (CARV) [0.1-100 micromol/l], an antihypertensive and cardiovascular drug with antioxidative properties, on superoxide generation (SO) and myeloperoxidase (MPO) release from isolated human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus. Unstimulated cells showed neither SO formation nor MPO release after preincubation with drug. CARV decreased fMLP and PMA stimulated MPO release and SO generation dose dependently. The inhibitory effect of CARV may attributed to non-specific action since its effect was not influenced by the type of stimulation. It might inhibit SO generation as well as MPO release either by membrane-operating stimulus (fMLP) or membrane bypassing activator (PMA).     

Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petunia x hybrida and Dendranthema x grandiflorum

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.     

Accumulation and translocation of Cd metal and the Cd-induced production of glutathione and phytochelatins in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

Translocation of cadmium (Cd) in the tissues of Vicia faba, the water content in biomass, the biomass production, and the glutathione and phytochelatin tissue concentrations were studied and correlated with the plant sensitivity and/or tolerance to Cd. The total concentrations of Cd were determined by inductively coupled plasma/mass spectrometry (ICP-MS), the concentrations of glutathione (GSH) and phytochelatins 2 and 3 (PC2 and PC3) were determined by on-line high performance liquid chromatography/electrospray-ionization tandem mass spectrometry (HPLC–ESI–MS–MS) in the roots and leaves of the sensitive and the tolerant cultivars of V. faba grown in Cd containing nutrient solutions (NS, 0–100 μmol l⁻¹ Cd²⁺). Both the cultivars of V. faba accumulate a major portion of Cd in the roots and only a minor part of ca. 4% in the leaves. The differences between the cultivars concerning Cd accumulation in leaves were apparent from higher Cd concentrations in NS and the Cd amount in the sensitive cultivar was approximately twice as high. In the roots, the differences between the cultivars in the Cd accumulation were only statistically significant with the highest Cd concentrations in NS, with the tolerant cultivar accumulating about 16% more of Cd compared to the sensitive one. The biomass production of the sensitive cultivar decreased approximately twice as fast with increasing Cd concentration in NS. The biomass water content decreased with increasing Cd concentration in NS in both the cultivars. In general, the GSH concentration did not linearly correlate with Cd accumulation, except for the roots of the sensitive cultivar where it was independent, and was higher in the sensitive cultivar than in the tolerant one in both the leaves and roots. The GSH concentration in leaves was approximately one order of magnitude higher than that in the roots for both the cultivars. The relationships between the PC and Cd concentrations in tissues were found nonlinear. At lower Cd accumulation levels, the PC concentrations followed an increase in the Cd accumulation in both the roots and leaves, whereas at higher Cd accumulations the relations differed between roots and leaves. In the roots, the PC concentrations decreased with increasing Cd accumulation, whereas the PC concentration in the leaves followed the decrease in the Cd accumulation.