Changes in chlorophyll a fluorescence, photosynthetic CO2 assimilation and xanthophyll cycle interconversions during dehydration in desiccation-tolerant and intolerant liverworts

The interactions among water content, chlorophyll a fluorescence emission, xanthophyll interconversions and net photosynthesis were analyzed during dehydration in desiccation-tolerant Frullania dilatata (L.) Dum. and desiccation-intolerant Pellia endiviifolia (Dicks) Dum. Water loss led to a progressive suppression of photosynthetic carbon assimilation in both species. Their chlorophyll fluorescence characteristics at low water content were: low photosynthetic quantum conversion efficiency, high excitation pressure on photosystem II and strong non-photochemical quenching. However, dissipation activity was lower in P. endiviifolia and was not accompanied by a rise in the concentration of de-epoxidised xanthophylls as F. dilatata. The photosynthetic apparatus of F. dilatata remained fully and speedily recuperable after desiccation in as indicated by the restoration of chlorophyll fluorescence parameters to pre-desiccation values upon rehydration. A lack of recovery upon remoistening of P. endiviifolia indicated permanent and irreversible damage to photosystem II. The results suggest that F. dilatata possesses a desiccation-induced zeaxanthin-mediated photoprotective mechanism which might aid photosynthesis recovery when favourable conditions are restored by alleviating photoinhibitory damage during desiccation. This avoidance mechanism might have evolved as an adaptative response to repeated cycles of desiccation and rehydration that represent a real threat to photosynthetic viability. Received: 12 January 1998 / Accepted: 14 July 1998     

Clovamide-rich extract from <Emphasis Type="Italic">Trifolium pallidum</Emphasis> reduces oxidative stress-induced damage to blood platelets and plasma

Numerous plants (including clovers) have been widely used in folk medicine for the treatment of different disorders. This in vitro study was designed to examine the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO⁻). Carbonyl groups and 3-nitrotyrosine in blood platelet and plasma proteins were determined by ELISA tests. Thiol groups level was estimated by using 5,5′-dithio-bis(2-nitro-benzoic acid, DTNB). Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric acid reactive substances. The results from our work indicate that clovamide-rich T. pallidum extract may reveal the protective properties in the prevention against oxidative stress. The presence of clovamide-rich T. pallidum extract (12.5–100 μg/ml) partly inhibited ONOO⁻-mediated protein carbonylation and nitration. All the used concentrations of T. pallidum extract reduced lipid peroxidation in plasma. The antioxidative action of the tested extract in the protection of blood platelet lipids was less effective; the extract at the lowest final concentration (12.5 μg/ml) had no protective effect against lipid peroxidation. The present results indicate that the extract from T. pallidum is likely to be a source of compounds with the antioxidative properties, useful in the prevention against the oxidative stress-related diseases.     

Manipulation of heterogeneity product in 4′-demethylepipodophyllotoxin biotransformation process by using yeast extract as nitrogen source

Manipulation of product heterogeneity was attempted by using yeast extract as nitrogen source in Alternaria alternata S-f6 transformation process of 4′-demethylepipodophyllotoxin. When the nitrogen source of NaNO₃ was replaced by yeast extract, the heterogeneity of biotransformation products was significantly varied from a single product (i.e., 4′-demethylpodophyllotoxone) to four podophyllum derivates. According to the kinetics of 4′-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6, the starting substrate of 4′-demethylepipodophyllotoxin was preferentially transformed to produce 4′-demethylpodophyllotoxone (1) with an oxidation reaction. By the further comparison of products configuration, 4β-caprinoyl-4′-demethylepipodophyllotoxin (3) was produced from 4′-demethylpodophyllotoxone (1) instead of 4′-demethylisopicropodophyllone (2), which might be produced from 4′-demethylpodophyllotoxone (1) with the isomerization of lactone. Finally, 4′-demethylisopicropodophyllone (2) was hydrolyzed to produce 3α-hydroxymethyl-(6, 7)-dioxol-4-one-naphthalene (4). This work shows new information on the 4′-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6 and provides a foundation for further studies on the structural diversification of a bioactive natural lead compound.     

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 μM), sodium nitroprusside (5 μM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1′-1′ Diphenyl-2′ picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.     

Effective Removal of Endocrine-Disrupting Compounds by Lignin Peroxidase from the White-Rot Fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624

The removal of endocrine-disrupting compounds (EDCs) by lignin peroxidase from white-rot fungus Phanerochaete sordida YK-624 (YK-LiP1) was investigated. Five endocrine disruptors, p–t-octylphenol (OP), bisphenol A (BPA), estrone (E₁), 17β-estradiol (E₂), and ethinylestradiol (EE₂) were eliminated by YK-LiP1 more effectively than lignin peroxidase from P. chrysosporium (Pc-LiP), and OP and BPA were disappeared almost completely in the reaction mixture containing YK-LiP1 after a 24-h treatment. Particularly, the removal of estrogenic activities of E₂ and EE₂, which show much higher estrogenic activities than other EDCs such as BPA and OP, were removed following 24-h treatment with YK-LiP1. Moreover, 5,5′-bis(1,1,3,3-tetramethylbutyl)-[1,1′-biphenyl]-2,2′-diol and 5,5′-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2′-diol were identified as the main metabolite from OP or BPA, respectively. These results suggest that YK-LiP1 is highly effective in removing of EDCs by the oxidative polymerization of these compounds.     

Hesperetin attenuates the highly reducing sugar-triggered inhibition of osteoblast differentiation

Diabetic bone disease is associated with increased oxidative damage and 2-deoxy-d-ribose (dRib) is used to induce oxidative damage similar to that observed in diabetics. To determine if hesperetin (3′,5,7-trihydroxy-4-methoxyflavanone) could influence osteoblast dysfunction induced by dRib, osteoblastic MC3T3-E1 cells were treated with dRib and hesperetin. Then, markers of osteoblast function and oxidative damage were examined. Hesperetin (10⁻⁷–10⁻⁵ M) caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and total antioxidant potential of MC3T3-E1 cells in the presence of 20 mM dRib (p < 0.05). Moreover, hesperetin (10⁻⁷ M) decreased cellular protein carbonyl (PCO), advanced oxidation protein products (AOPP), and malondialdehyde (MDA) contents of osteoblastic MC3T3-E1 cells in the presence of 20 mM dRib. These results demonstrate that hesperetin attenuates dRib-induced damage, suggesting that hesperetin may be a useful dietary supplement for minimizing oxidative injury in diabetes related bone diseases.     

Phenotypic plasticity and biomass allocation pattern in three <Emphasis Type="Italic">Dryopteris</Emphasis> (Dryopteridaceae) species on an experimental light-availability gradient

We were interested in whether the contrasting regional distribution patterns of three congeneric, frequently co-occurring fern species (Dryopteris carthusiana, D. dilatata and D. expansa) could be explained by differential biomass allocation strategies and different phenotypic plasticities to light availability. The morphology and habitat preference of these ferns are known to be very similar, but in Estonia, their frequencies of occurrence differ sharply––Dryopteris carthusiana is common, D. expansa grows in scattered localities, and D. dilatata is rare. We grew the species under different levels of illumination (100, 50, 25 and 10% of full daylight) in an experimental garden to compare their autecological responses to shading. After one growing season there were clear interspecific differences in total plant biomass accumulation––D. carthusiana > D. expansa > D. dilatata––indicating the possible competitive inferiority of the latter at the young sporophyte stage. D. expansa was the least shade-tolerant, with biomass decreasing sharply under less than 50% illumination; D. dilatata was the most shade-tolerant, with similar growth at all illumination levels. In relative biomass allocation patterns, the most notable differences among species were in the relative shares of biomass stored in rhizomes. In D. carthusiana and D. expansa this share was nearly constant and independent of the illumination conditions. D. dilatata allocated very little biomass into rhizome in deep shade, but was able to increase this share more than twofold in 50% light. Dryopteris dilatata was clearly shown to be morphologically the most plastic of the three. In four traits––rhizome mass, frond:below-ground biomass ratio, stipe length and specific leaf area––its degree of ontogenetic plasticity to light was significantly higher than that of D. expansa and D. carthusiana. While the general performance (biomass production) of species in the experiment coincided with that observed in nature, the results of plasticity estimation were somewhat surprising––it is difficult to explain the inferior performance of a species (D. dilatata) through high morphological plasticity. Probably, the species is rare either because of certain climatic restrictions, or because it is presently expanding its distribution and is in the phase of invading Estonian understory communities.     

Red cabbage anthocyanins may protect blood plasma proteins and lipids

The present in vitro study was designed to examine the antioxidative activity of red cabbage anthocyanins (ATH) in the protection of blood plasma proteins and lipids against damage induced by oxidative stress. Fresh leaves of red cabbage were extracted with a mixture of methanol/distilled water/0.01% HCl (MeOH/H₂O/HCl, 50/50/1, v/v/w). Total ATH concentration [μM] was determined with cyanidin 3-glucoside as a standard. Phenolic profiles in the crude red cabbage extract were determined using the HPLC method. Plasma samples were exposed to 100 μM peroxynitrite (ONOO⁻) or 2 mM hydrogen peroxide (H₂O₂) in the presence/absence of ATH extract (5–15 μM); oxidative alterations were then assessed. Pre-incubation of plasma with ATH extract partly reduced oxidative stress in plasma proteins and lipids. Dose-dependent reduction of both ONOO⁻ and H₂O₂-mediated plasma protein carbonylation was observed. ATH extract partly inhibited the nitrative action of ONOO⁻, and significantly decreased plasma lipid peroxidation caused by ONOO⁻ or H₂O₂. Our results demonstrate that anthocyanins present in red cabbage have inhibitory effects on ONOO⁻ and H₂O₂-induced oxidative stress in blood plasma components. We suggest that red cabbage ATH, as dietary antioxidants, should be considered as potentially usable nutraceuticals in the prevention of oxidative stress-related diseases.     

Structure,function and biosynthesis of carotenoids in the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus halophilus</Emphasis>

Inhibitor studies and mutant analysis revealed a C₃₀ pathway via 4,4′-diapophytoene and 4,4′-diaponeurosporene to 4,4′-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4′-diapophytoene synthase and a 4,4′-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4′-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4′-diaponeurosporene and in an orange mutant at the stage of 4,4′-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.     

Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes

Arsenic is a naturally occurring element that is present in food, soil, and water. Inorganic arsenic can accumulate in human skin and is associated with increased risk of skin cancer. Oxidative stress due to arsenic exposure is proposed as one potential mode of carcinogenic action. The purpose of this study is to investigate the specific reactive oxygen and nitrogen species that are responsible for the arsenic-induced oxidative damage to DNA and protein. Our results demonstrated that exposure of human keratinocytes to trivalent arsenite caused the generation of 8-hydroxyl-2′-deoxyguanine (8-OHdG) and 3-nitrotyrosine (3-NT) in a concentration- and time-dependent manner. Pentavalent arsenate had similar effects, but to a significantly less extent. The observed oxidative damage can be suppressed by pre-treating cells with specific antioxidants. Furthermore, we found that pre-treating cells with Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), or with 5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride (FeTMPyP), a decomposition catalyst of peroxynitrite, suppressed the generation of both 8-OHdG and 3-NT, which indicated that peroxynitrite, a product of the reaction of nitric oxide and superoxide, played an important role in arsenic-induced oxidative damage to both DNA and protein. These findings highlight the involvement of peroxynitrite in the molecular mechanism underlying arsenic-induced human skin carcinogenesis.     

Protein cysteine <Emphasis Type="Italic">S</Emphasis>-guanylation and electrophilic signal transduction by endogenous nitro-nucleotides

Nitric oxide (NO), a gaseous free radical that is synthesized in organisms by nitric oxide synthases, participates in a critical fashion in the regulation of diverse physiological functions such as vascular and neuronal signal transduction, host defense, and cell death regulation. Two major pathways of NO signaling involve production of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and posttranslational modification (PTM) of redox-sensitive cysteine thiols of proteins. We recently clarified the physiological formation of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) as the first demonstration, since the discovery of cGMP more than 40 years ago, of a new second messenger derived from cGMP in mammals. 8-Nitro-cGMP is electrophilic and reacts efficiently with sulfhydryls of proteins to produce a novel PTM via cGMP adduction, a process that we named protein S-guanylation. 8-Nitro-cGMP may regulate electrophilic signaling on the basis of its electrophilicity through induction of S-guanylation of redox sensor proteins. Examples include S-guanylation of the redox sensor protein Kelch-like ECH-associated protein 1 (Keap1), which leads to activation of NF-E2-related factor 2 (Nrf2)-dependent expression of antioxidant and cytoprotective genes. This S-guanylation-mediated activation of an antioxidant adaptive response may play an important role in cytoprotection during bacterial infections and oxidative stress. Identification of new redox-sensitive proteins as targets for S-guanylation may help development of novel therapeutics for oxidative stress- and inflammation-related disorders and vascular diseases as well as understanding of cellular protection against oxidative stress.     

Oligonucleotide analogues containing a C3′-NH-C(O)-CH<Subscript>2</Subscript>-C5′ amide internucleotide bond

A dinucleotide containing a C3′-NH-C(O)-CH₂-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′-O-(tert-butyldimethylsilyl)thymidine with 3′-O-benzyl-2′-O-tert-butyldimethylsilyl-5′-deoxy-5′-carboxymethylribosylthymine, which was obtained from 2′-O-acetyl-3′-O-benzyl-5′-deoxy-5′-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protecting groups, the dinucleotide was converted into 3′-O(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones.     

Role of Spectral Studies in Detection of Antibacterial Phytoelements and Phytochemicals of Moringa oleifera

This paper reports, the Laser Induced Breakdown Spectroscopy (LIBS) studies and structure elucidation of compounds isolated from the fruit extract of Moringa oleifera and also deals with their possible effects on some bacterial strains viz. Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa. The extract was found to be active against all four microorganisms used. Extent of inhibitory effect of extract was assessed at different concentrations of 25, 50, 75 mg/ml by measuring diameter of inhibition zone (DIZ). Our results clearly showed that the 75 mg/ml concentration of the extract had 14, 12 and 18 mm of the DIZ against Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa and 14 mm with 50 mg/ml concentration against Escherichia coli. The results were compared with the standard antibiotic ‘ampicillin’ of 1 mg/ml concentration. LIBS was recorded with high power pulsed laser beam from Nd: YAG Laser (Continuum Surelite III-10), focused on the surface of the material, which was in liquid form, to generate plasma on the surface of the sample. LIBS data clearly demonstrate the presence of trace elements, magnesium and iron, in high concentration in the extract. Whereas, from the phytochemical profile reveals the presence of two new compounds, S-ethyl-N-{4-[(α-L-rhamnosyloxy) benzyl]} thiocarbamate and 2-acetoxy {4-[(2′,3′,4′-tri-O-acetyl-α-L-rhamnosyloxy) benzyl]} acetonitrile as the major constituents. This study is the first report on synergetic effect of the phytoconstituents and certain set of elements present in their defined role in bacterial management against different bacterial strains.     

Identification of IS elements in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains grown in a medium with ferrous iron or adapted to elemental sulfur

IS elements were identified in the genomes of five Acidithiobacillus ferrooxidans strains isolated from various media. IST2 elements were revealed in all the strains grown in a medium with ferrous iron, ISAfe1 elements were detected in four strains (TFBk, TFL-2, TFV-1 and TFO). Three strains (TFV-1, TFN-d and TFO) were found to contain IS elements, ~600 bp long. These were named preliminary as ISAfe600. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of an ISAfe1 element in TFBk and TFL-2 strains and complete sequencing of the ISAfe1 element in the TFBk strain has revealed nucleotide substitutions as compared to the prototype, i.e., the ISAfe1 element of an ATCC 19859 strain. Partial sequencing of the 5′- and 3′-terminal nucleotide stretches of the IST2 elements in TFO, TFBk and TFL-2 strains has shown numerous nucleotide substitutions when compared to the IST2 element of an ATCC 19859 strain. Complete sequencing of the IST2 element in the TFBk strain has revealed: the divergence between the IST2 elements in the TFBk strain and the prototype was 21.2%. Southern hybridization of EcoRI fragments of the chromosomal DNA from five A. ferrooxidans strains grown in a medium with ferrous iron using an internal region of ISAfe1, a full-length ISAfe1 or a full-length IST2 as probes has shown them to differ in the number of copies of IS elements and their localization on the chromosomes. Adaptation to elemental sulfur in A. ferrooxidans strains caused changes in the number, intensity and localization of hybridization bands. The authors discuss the role of IS elements in the adaptation of A. ferrooxidans to the new energy substrate. The nucleotide sequence data reported in this paper were deposited in GenBank under accession numbers: AY823401, the ISAfe1 from A. ferrooxidans TFBk; AY825254, the IST2 from TFBk; DQ002894, the 5′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ002895, the 3′-terminal nucleotide sequence of ISAfe1 from TFL-2; DQ005952, the 5′-terminal nucleotide sequence of IST2 from TFV-1; DQ005953, the 3′-terminal nucleotide sequence of IST2 from TFV-1.     

Decolorization of 1-aminoanthraquinone-2-sulfonic acid by Sphingomonas xenophaga

Sphingomonas xenophaga QYY from sludge samples could effectively decolorize 1-aminoanthraquinone-2-sulfonic acid (ASA-2), one kind of anthraquinone dye intermediate, under aerobic conditions. More than 98% of ASA-2 could be removed within 120 h at the dye concentration from 200 mg l⁻¹ to 1,000 mg l⁻¹ due to oxidative degradation. The strain converted ASA-2 to 2-(2′-hydroxy-3′-amino-4′-sulfo-benzoyl)-benzoic acid, 2-(2′-amino-3′-sulfo-6′-hydroxy-benzoyl)-benzoic acid, o-phthalic acid and 2-amino-3-hydroxy-benzenesulfonic acid, which were identified using HPLC-MS and NMR. A possible initial decolorization pathway was proposed according to these metabolites. The decolorization of ASA-2 by cells in the basal salt medium was induced by ASA-2, and was due to soluble cytosolic enzymes. Combined initial decolorization pathway and the analysis of decolorization enzyme(s), the major enzyme responsible for ASA-2 decolorization was a NADH-dependent oxygenase.     

Protective effect of Raphanus sativus on H2O2 induced oxidative damage in human lymphocytes

Raphanus sativus, a common cruciferous vegetable has been attributed to possess a number of pharmacological and therapeutic properties. This present study evaluated the protective effect of different parts of R. sativus such as root, stem and leaf obtained with a variety of extraction solvents against cell death and oxidative DNA damage induced by hydrogen peroxide (H₂O₂) in normal human lymphocytes. R. sativus extracts as such showed no cytotoxicity and genotoxicity to the lymphocytes at the tested concentrations. Of the different extracts, hexane extract of root and methanolic extract of stem and leaf showed significant protective effect against oxidative damage induced by 200 μM H₂O₂ in a dose dependent manner, as compared to cells exposed only to H₂O₂. Our results suggest that the protective effect afforded by R. sativus extract could be related to the presence of isothiocyanates and polyphenolics, as they possess significant capacity to remove reactive species by virtue of their ability to scavenge free radicals and induce antioxidant enzyme system in the cells.     

Extract from <Emphasis Type="Italic">Conyza canadensis</Emphasis> as a modulator of plasma protein oxidation induced by peroxynitrite <Emphasis Type="Italic">in vitro</Emphasis>

The antioxidative activity of the extract from Conyza canadensis in plasma treated with peroxynitrite (ONOO⁻) (0.1 mM) was studied. C. canadensis is known to possess a broad set of pharmacological effects because of content of various antioxidants, antiplatelet and anticoagulant compounds. The aim of our study was to assess if this extract protects plasma proteins against oxidative/nitrative damages induced by ONOO⁻. The plasma components are continuously exposed to reactive oxygen/nitrogen species action. Peroxynitrite evokes oxidative stress and induces undesirable effects in biological systems and causes damage to biomolecules. The extract from Conyza (50–2500 mg/ml) caused a dose-dependent reduction of protein nitration by 90%. The oxidation of plasma proteins was diminished by about 75%. ONOO⁻ oxidized the plasma thiol groups and this process was inhibited by tested extract. The level of reduced protein thiols was increased thrice at the lowest concentration of extract (50 mg/ml). The highest concentration of extract decreased twice the level of protein thiols in reduced forms and increased the homocysteine level about 4.5 times. The obtained results demonstrated that the extract from Conyza possesses antioxidative properties in vitro, protects plasma proteins against toxicity induced by peroxynitrite and has modulating effects on thiol/disulfide redox status.     

Antioxidant enzyme inhibitors enhance peroxynitrite-induced cell death in U937 cells

Peroxynitrite, a potent physiological inorganic toxin, is known to play a critical role in cellular oxidative damage. The protective role of antioxidant enzymes against peroxynitrite-induced oxidative damage in U937 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole, and oxlalomalate, specific inhibitors of superoxide dismutase, catalase, and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to 1 mM 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion, to U937 cells, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage reflected by an increase in 8-hydroxy-2′-deoxyguanosine, were higher in the inhibitor-treated cells as compared to the control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2′7′-dichlorodihydrofluorescin as well as the significant decrease in the intracellular GSH level in the inhibitor-treated U937 cells upon exposure to SIN-1. These results suggest that antioxidant enzymes play an important role in cellular defense against peroxynitrite-induced cell death.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.