Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Hosts and distribution of Viscum album L. ssp. album in Croatia and Slovenia

Abstract

Viscum album L. ssp. album is semi-parasitic on deciduous trees and shrubs. In order to identify hosts and map the distribution of V. album ssp. album in Croatia and Slovenia, field research was carried out, and herbaria were surveyed. In Croatia and Slovenia, V. album ssp. album occurred on 59 taxa. In Croatia, there were 52 hosts (33 autochthonous and 15 allochthonous species, two cultivars and two hybrids). In Slovenia, there were 25 hosts (21 autochthonous and four allochthonous species). There were 18 hosts common to both countries, 34 hosts were found only in Croatia, and seven hosts only in Slovenia. The hosts belonged to 13 families. The majority of these (19 species) belong to the Rosaceae, followed by Salicaceae, Aceraceae, Betulaceae, Fagaceae, Juglandaceae, Tiliaceae, Hippocastanaceae, Ulmaceae, Oleaceae, Fabaceae, Moraceae and Viscaceae. All hosts have been previously recorded in the literature, except Alnus japonica (Thunb.) Steud., Amelanchier lamarckii F.G. Schroed. and Crataegus nigra Waldst. et Kit. The distribution of this mistletoe was scattered, due to the scattered distribution of hosts, local conditions, movement of bird-vectors, etc. A continuous distribution was found only in part of the distribution area of narrow-leaved ash (Fraxinus angustifolia Vahl).     

Leaflet morphometric variation of service tree (Sorbus domestica L.) in the Balkan Peninsula

Abstract

In most European countries, the service tree (Sorbus domestica L.) is a rare and threatened species and its conservation has been recognised as a priority. The aim of this study was to asses its morphologic variation in the western and central part of the Balkan Peninsula and in southern Central Europe. Three populations were analysed: one in Serbia, one in Bosnia and Herzegovina and one in Slovenia. In each population 30 trees were selected, and from each tree 30 leaves were collected for morphometric analysis based on nine leaflet morphological traits. Univariate (ANOVA) and multivariate (MANOVA) analysis of variance were used to estimate the variation within- and between populations and a discriminant analysis was performed to examine the structure of the between-population differences. The values of particular morphological traits found in our study did not differ considerably from the values reported elsewhere. The results revealed significant within- and between population variation. Variation within populations was highly significant for all the scored leaf morphological traits, while variation between populations was significant for all the studied traits except for the leaflet length. The discrimination between the three populations was significant. High percentages of correctly classified samples demonstrate good discriminating employability of the analysed leaf morphological traits and indicate differentiation of the analysed populations.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Design,synthesis and cholinesterase inhibitory properties of new oxazole benzylamine derivatives

Abstract

The enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are primary targets in attenuating the symptoms of neurodegenerative diseases. Their inhibition results in elevated concentrations of the neurotransmitter acetylcholine which supports communication among nerve cells. It was previously shown for trans-4/5-arylethenyloxazole compounds to have moderate AChE and BChE inhibitory properties. A preliminary docking study showed that elongating oxazole molecules and adding a new NH group could make them more prone to bind to the active site of both enzymes. Therefore, new trans-amino-4-/5-arylethenyl-oxazoles were designed and synthesised by the Buchwald-Hartwig amination of a previously synthesised trans-chloro-arylethenyloxazole derivative. Additionally, naphthoxazole benzylamine photoproducts were obtained by efficient photochemical electrocyclization reaction. Novel compounds were tested as inhibitors of both AChE and BChE. All of the compounds exhibited binding preference for BChE over AChE, especially for trans-amino-4-/5-arylethenyl-oxazole derivatives which inhibited BChE potently (IC₅₀ in µM range) and AChE poorly (IC₅₀?100?µM). Therefore, due to the selectivity of all of the tested compounds for binding to BChE, these compounds could be applied for further development of cholinesterase selective inhibitors.

• HIGHLIGHTS

• Series of oxazole benzylamines were designed and synthesised

• The tested compounds showed binding selectivity for BChE

• Naphthoxazoles were more potent AChE inhibitors

     

The Novel 6–(N-Pyrrolyl)purine Acyclic Nucleosides: 1H and 13C NMR and X-ray Structural Study†

Abstract

Synthesis of the novel nucleoside analogues containing exocyclic pyrrolo moiety and acyclic side chains attached to the purine ring at N-9 and N-7 is described. The site of alkylation was determined by ¹H and ¹³C NMR on the basis of chemical shifts, C-H coupling constants and connectivity in NOESY and HETCOR spectra. The N-9 substitution of 7 was proved by its X-ray crystallographic analysis.

     

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1–3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4–8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and label-free quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS)¹ has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9–12). On the other hand, label-free quantitation has gained momentum in recent years (13–15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quantitation. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides. Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples (20). One solution to large-scale quantitation without any isotope labeling is pseudo internal standard approach (21), which selects endogenous house-keeping proteins as the internal standard so that no spike-in reagent is required. However, finding a good pseudo internal standard remains a challenge for phosphoproteome samples. Spike-in experiments are an alternative way to improve normalization profile. Some internal standard peptides such as MassPREP^TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22–24). There are two major normalization procedures. In one approach, sample peptides were normalized to the total peak intensity of spike-in peptides (25). Alternatively, the digested peptides were compared at first and the normalization factor was determined in different ways such as the median (26) or average of ratios (27). However, spiking an identical amount of standard proteins into phosphoproteomic samples before protein digestion may not be compatible with phosphoproteomic analyses which typically require a phosphopeptide enrichment step. Spectral counting has been extensively applied in large sets of proteomic samples because of its simplicity but the method is often not reliable for the quantitation of phosphoproteins, which are typically identified by single phosphopeptides with few spectra (28–30). Many software packages have been implemented to support the wide variety of those quantitation techniques, including commercial platforms such as Progenesis LC-MS^TM, Mascot Distiller^TM, PEAKS Q^TM, etc., as well as open-source software packages including MaxQuant (31), PEPPeR (32), Skyline (33), etc.In this study, we have devised a novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency (Fig. 1). The approach employs synthetic peptide libraries as the internal standard. These peptides were prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides were designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity is performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (34). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide.Open in a separate windowFig. 1.Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R²) and accuracy (%Acc).Water deficit and salinity causes osmotic stress, which is a major environmental factor limiting plant agricultural productivity. Osmotic stress rapidly changes the metabolism, gene expression and development of plant cells by activating several signaling pathways. Several protein kinases have been characterized as key components in osmotic stress signaling. Arabidopsis histidine kinase AHK1 can complement the histidine kinase mutant yeast, which can act as the osmosensor in yeast (35). Overexpression of AHK1 gene increases the drought tolerance of transgenic plants in Arabidopsis (36). Similar to yeast, the MAPK kinase cascade is also involved in osmotic stress response in plants. It is reported that AtMPK3, AtMPK6, and tobacco SIPK can be activated by NaCl or mannitol, and play positive roles in osmotic signaling (37, 38). MKK7 and MKKK20 may act as the up-stream kinase in the kinase cascade (39). Involvement of some calcium-dependent protein kinases, such as AtCPK21, AtCPK6, and OsCPK7 (CDPK) in osmotic stress signaling has also been reported (40–42). Another kinase family, SNF1-related protein kinase (SnRK) 2, also participates in osmotic stress response. In Arabidopsis, there are ten members in the SnRK2 family. Five from the ten SnRK2s, SnRK2, 3, 6, 7, and 8, can be activated by abscisic acid (ABA) and play central roles in ABA-receptor coupled signaling (43, 44). Furthermore, all SnRK2s except SnRK2.9 can be activated by NaCl or mannitol treatment (43). The decuple mutant of SnRK2 showed a strong osmotic hypersensitive phenotype (45). It is proposed that protein kinases including MAPK and SnRK2s have a critical function in osmotic stress (46), but the detailed mechanism and downstream substrates or target signal components are not yet clarified. We applied, therefore, the LAXIC approach with a self-validating method (47) to profile the osmotic stress-dependent phosphoproteome in Arabidopsis by quantifying phosphorylation events before and after mannitol treatment. Among a total of over 2000 phosphopeptides, more than 400 of them are dependent on osmotic stress. Those phosphoproteins are present on enzymes participating in signaling networks that are involved in many processes such as signal transduction, cytoskeleton development, and apoptosis. Overall, LAXIC represents a powerful tool for label-free quantitative phosphoproteomics.     

Chemical Diversity of the Natural Populations of Dalmatian Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) in Croatia

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir. ) Sch.Bip. ) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households. A total of 25 Dalmatian pyrethrum populations from Croatia were studied to determine the pyrethrin content and composition, and to identify distinct chemotypes. The total pyrethrin content ranged from 0.36 to 1.30% (dry flower weight; DW) and the pyrethrin I/pyrethrin II ratio ranged from 0.64 to 3.33%. The statistical analyses revealed that the correlations between the percentage of pyrethrin I and of all the other components were significant and negative. The total pyrethrin content was positively correlated with the percentage of pyrethrin I and negatively correlated with cinerin II. The multivariate analysis of the chemical variability enabled the identification of five chemotypes among 25 Dalmatian pyrethrum populations. The chemical characterization of indigenous Dalmatian pyrethrum populations may serve as a good background for future breeding and agricultural exploitation.     

Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

Variability of the Needle Essential Oils of Juniperus deltoides R.P.Adams from Different Populations in Serbia and Croatia

The essential‐oil compositions of one Croatian and three Serbian populations of Juniperus deltoides R.P.Adams have been determined by GC/MS analysis. In total, 147 compounds were identified, representing 97.3–98.3% of the oil composition. The oils were dominated by monoterpenes, which are characteristic components for the species of the section Juniperus. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, with a summed‐up average content of 49.45%. Statistical methods were used to determine the diversity of the terpene classes and the common terpenes between the newly described J. deltoides populations from Serbia and Croatia. Only reports on several specimens from this species have been reported so far, and there are no studies that treat population diversity. Cluster analysis of the oil contents of 21 terpenes showed high correlation with the geographical distribution of the populations, separating the Croatian from the Serbian populations. The comparison of the essential‐oil compositions obtained in the present study with literature data, showed the separation of J. deltoides and J. oxycedrus ssp. oxycedrus from the western Mediterranean region.