Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.     

Codeine and 6-Acetylcodeine Analgesia in Mice

Summary 1. Acetylation of morphine at the 6-position changes its pharmacology. To see if similar changes are seen with codeine, we examined the analgesic actions of codeine and 6-acetylcodeine.2. Like codeine, 6-acetylcodeine is an effective analgesic systemically, supraspinally and spinally, with a potency approximately a third that of codeine.3. The sensitivity of 6-acetylcodeine analgesia to the mu-selective antagonists β-FNA and naloxonazine confirmed its classification as a mu opioid. However, it differed from the other mu analgesics in other paradigms.4. Antisense mapping revealed the sensitivity of 6-acetylcodeine to probes targeting exons 1 and 2 of the mu opioid receptor gene (Oprm), a profile distinct from either codeine or morphine. Although heroin analgesia also is sensitive to antisense targeting exons 1 and 2, heroin analgesia also is sensitive to the antagonist 3-O-methylnaltrexone, while 6-acetylcodeine analgesia is not.5. Thus, 6-acetylcodeine is an effective mu opioid analgesic with a distinct pharmacological profile.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

Lipocalins - a family portrait

Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through membrane-localized protein receptors.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP(+) oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.Electronic supplementary material The online version of this article (doi:10.1007/s10867-012-9262-z) contains supplementary material, which is available to authorized users.     

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.     

EPR study of thylakoid membrane dynamics in mutants of the carotenoid biosynthesis pathway of Synechocystis sp. PCC6803

EPR spectroscopy using 5-doxylstearic acid (5-SASL) and 16-doxylstearic acid (16-SASL) spin probes was used to study the fluidity of thylakoid membranes. These were isolated from wild type Synechocystis and from several mutants in genes encoding selected enzymes of the carotenoid biosynthesis pathway and/or acyl-lipid desaturases. Cyanobacteria were cultivated at 25°C and 35°C under different light regimes: photoautotrophically (PAG) and/or in light-activated heterotrophic conditions (LAHG). The relative fluidity of membranes was estimated from EPR spectra based on the empirical outermost splitting parameter in a temperature range from 15°C to 40°C. Our findings demonstrate that in native thylakoid membranes the elimination of xanthophylls decreased fluidity in the inner membrane region under optimal growth conditions (25°C) and increased it under sublethal heat stress (35°C). This indicated that the overall fluidity of native photosynthetic membranes in cyanobacteria may be influenced by the ratio of polar to non-polar carotenoid pools under different environmental conditions.     

Slow vacuolar channels in vacuoles from winter and spring varieties of rape (Brassica napus)

Currents passing through slowly activating vacuolar channels (SV) in isolated vacuoles from winter (Górczański) and spring (M?ochowski) varieties of rape (Brassica napus) were examined using the patch-clamp technique. Eight-week-long vernalization at 5/2 degrees C (day/night) was applied to obtain the generative stage of winter rape. SV channels of vacuoles isolated from vegetative (rosette) and generative leaves of both varieties were examined in order to investigate a possible role of these ion channels in rape flowering. Single SV channel conductance measured in a vacuole-attached configuration (natural cell sap) ranged from 60 to 83 pS. Lower values were observed in the generative leaves of both varieties. Unitary conductance measured in excised cytoplasm-out membrane patches did not differ significantly among the experimental variants, with the exception of spring generative vacuoles, where it was significantly lower. There was also no difference in SV current densities measured in the whole-vacuole configuration. Gibberellic acid (GA(3)) (2mg/l) caused lowering of macroscopic SV currents by 20%, and had no significant effect on the single channel conductance. We conclude that SV channels play a role in rape vernalization and flowering owing to their multifactor regulation abilities rather than structural changes.