Expression of an anther-specific chalcone synthase-like gene is correlated with uninucleate microspore development in Nicotiana sylvestris

Two cDNA clones, specifically expressed in Nicotiana sylvestris anthers during uninucleate microspore development, were isolated using a subtractive hybridization approach. Sequence analysis showed that one of them, NSCHSLK, displayed a high level of similarity to several anther-specific chalcone synthase-like (CHSLK) proteins and an ORF from chromosome 1 of Arabidopsis thaliana. A lower, but significant, similarity to chalcone synthases and closely related enzymes (CHSRE) was also detected. The structure of the nschslk gene was found to be typical of the chalcone (chs) / stilbene (sts) synthase family. Expression of NSCHSLK mRNA was confined to microspores and tapetal cells. UV-irradiation or infection with Phytophthora parasitica var. nicotianae of transgenic Nicotiana benthamiana plants carrying a chimeric nschslk/GUS gene indicated that the nschslk promoter exhibits the same anther-specific, developmentally regulated expression pattern. Comparison of CHSRE and CHSLK polypeptide sequences revealed some important similarities and differences between the two groups. The data presented in this study, suggest that the anther-specific chslk genes represent a separate sub-family of plant polyketide synthases related to chs/sts in terms of gene structure, polypeptide sequence and the possible catalytic mechanism, but differing in substrate/product specificity. The putative role of CHSLK enzymes in anther development and particularly in exine synthesis is discussed.     

Thiol antidotes effect on lipid peroxidation in mercury-poisoned rats

The effect of thiol antidotes 2,3-dimercapto-1-propanesulfonic acid (DMPS) and D-penicillamine (PA) on lipid peroxidation and on activities of some protecting enzymes in blood, liver and kidneys of mercury-poisoned rats has been studied. It has been found that Hg-poisoning is associated with increased lipid peroxidation in the liver and in the kidneys and with inactivation of superoxide dismutase (SOD) and catalase. Inhibition of SOD is caused by thiols treatment too, but in this case acceleration of lipid peroxidation has not been observed. Evidence is presented that in the liver, protection against mercury-induced lipid peroxidation is afforded by both thiols, while in the kidneys only PA has protective effect. In in vitro experiments it has been demonstrated that both antidotes can act as O2- scavengers and lipid peroxidation inhibitors, but PA is significantly more effective. On the basis of the obtained results a conclusion is drawn that in addition to the metal-removing ability, the antioxidant properties of the chelating agents may play an important role in manifestation of their beneficial effect in metal intoxications.     

Relationships between structure of 5-nitro-2-furylethylenes and their SOS-function-inducing activities in Escherichia coli

The SOS-function-inducing activities of 36 furylethylenes were characterized in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ 37. To correct for the inhibitory effects of test compounds on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Tested furylethylenes included nine alkylesters and eleven N-alkylamides of 5-nitro-2-furylacrylic acid (NFAA) and fourteen derivatives differing not only in substituents at exocyclic double bond, but also in the position 5 of the furan ring. The induction of the SOS-function by the derivatives depends on the presence of 5-nitrofuran centre in their molecule; side chains in the position 2 modify the degree of SOS response. SOS-inducing potency of n-alkyl congeners decreases with increasing lipophilicity. Effect of derivatives with branched alkyl substituents is lower than expected from the behavior of the n-alkyl homologues. All derivatives with positive effect on SOS-function in E. coli show mutagenic activity on Salmonella typhimurium TA98 in Ames test.     

Relationship between the hemolytic action of heavy metals and lipid peroxidation

It is well known that some of the heavy metals have a hemolytic action, but the mechanisms responsible for this effect are not well established. In order to elucidate whether or not the hemolytic action of heavy metal ions is associated with the peroxidation of membrane lipids, the relationship between metal-induced hemolysis and the generation of malonaldehyde has been studied.The results obtained show that metal-induced hemolysis is associated with the development of peroxidative processes in erythrocyte membranes. The peroxidation is caused by metals with and without pro-oxidant catalytic action. The level of the malonaldehyde products rises before the appearance of hemolysis which proves that the development of peroxidative processes precedes but does not result from hemolysis.The suggestion has been made that the peroxidation of membrane lipids is a possible mechanism of damage to the red cell membrane in metal-induced hemolysis.     

Targeting Mitochondria by Zn(II)N-Alkylpyridylporphyrins: The Impact of Compound Sub-Mitochondrial Partition on Cell Respiration and Overall Photodynamic Efficacy

Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.     

Possible contribution of oxyhemoglobin to the iron-induced hemolysis simultaneous effect of iron and hemoglobin on lipid peroxidation

The mechanism of iron toxicity in iron overloaded patients is not well established. A hypothesis was put forward that free radical processes are involved. Our earlier study indicates that iron-induced hemolysis is preceded by peroxidation of the membrane lipids. In the present work the simultaneous effect of iron and hemoglobin on lipid peroxidation was studied. It was found that in hemoglobin-containing liposome suspensions Fe2+ in concentrations above 10(-5) M inhibits the peroxidation, while Fe3+ drastically potentiates it, with concomitant transformation of oxyhemoglobin to methemoglobin. The experiments with scavengers of activated oxygen indicate superoxide anion radical (O-.2), hydroxyl radical (OH.) and singlet oxygen (1O2) participation. The possible mechanism of the phenomenon is discussed. A conclusion is drawn that the toxic effect of Fe3+ may be associated not only with iron--membrane interaction, but also with increased methemoglobin formation and O-.2 release.     

Photosensitizing action of isomeric zinc N-methylpyridylporphyrins in human carcinoma cells

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N-alkylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺ and ZnTE-2-PyP⁴⁺) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP⁴⁺ (ZnTM-2(3,4)-PyP⁴⁺) and respective ligands on a human colon adenocarcinoma cell line. At 10 μM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 μM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP⁴⁺ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD).