Electrophysiology of respiratory chain complexes and the ADP-ATP exchanger in native mitochondrial membranes

Transport of protons and solutes across mitochondrial membranes is essential for many physiological processes. However, neither the proton-pumping respiratory chain complexes nor the mitochondrial secondary active solute transport proteins have been characterized electrophysiologically in their native environment. In this study, solid-supported membrane (SSM) technology was applied for electrical measurements of respiratory chain complexes CI, CII, CIII, and CIV, the F(O)F(1)-ATPase/synthase (CV), and the adenine nucleotide translocase (ANT) in inner membranes of pig heart mitochondria. Specific substrates and inhibitors were used to validate the different assays, and the corresponding K(0.5) and IC(50) values were in good agreement with previously published results obtained with other methods. In combined measurements of CI-CV, it was possible to detect oxidative phosphorylation (OXPHOS), to measure differential effects of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the respective protein activities, and to determine the corresponding IC(50) values. Moreover, the measurements revealed a tight functional coupling of CI and CIII. Coenzyme Q (CoQ) analogues decylubiquinone (DBQ) and idebenone (Ide) stimulated the CII- and CIII-specific electrical currents but had inverse effects on CI-CIII activity. In summary, the results describe the electrophysiological and pharmacological properties of respiratory chain complexes, OXPHOS, and ANT in native mitochondrial membranes and demonstrate that SSM-based electrophysiology provides new insights into a complex molecular mechanism of the respiratory chain and the associated transport proteins. Besides, the SSM-based approach is suited for highly sensitive and specific testing of diverse respiratory chain modulators such as inhibitors, CoQ analogues, and uncoupling agents.     

Seed consumption and dispersal of ant-dispersed plants by slugs

In beech-dominated forests in Central Europe, many spring geophytes show adaptations to seed dispersal by ants (myrmecochory). Ants, however, can be rare in such moist forests. Motivated by observations of slug feeding on seeds we investigated the seed consumption of two plant species, Anemone nemorosa and Asarum europaeum, by slugs, in a series of experiments. In a seed predation experiment in a beech forest, we found that seed removal was strongly reduced when gastropods were excluded from the seed depots. The contribution of insects, including ants, and rodents to seed removal was relatively less but differed between May and July. In the laboratory, slug species, in particular Arion sp., consumed seeds of both plant species. Slugs either consumed the elaiosomes of seeds or swallowed seeds intact. Swallowed seeds were defecated undamaged and germinated as well as control seeds when buried overwinter, indicating the potential for seed dispersal by slugs. We also recovered seeds of myrmecochores in the faeces of several slugs caught in forests. In a slug release experiment in the forest, slugs moved up to 14.6 m (mean 4.4 m) in 15 h, which is the median gut passage time of seeds based on measurements made in the laboratory. We also found that when slug-defecated seeds were offered to rodents, these were less attractive than control seeds, suggesting that passage through the slug gut reduces seed predation risk. Our results demonstrate that slugs are significant consumers of elaiosomes or entire seeds of ant-dispersed plants and that they can function as seed dispersers of these plants.     

Differential Impact of Lipoxygenase 2 and Jasmonates on Natural and Stress-Induced Senescence in Arabidopsis

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.Senescence is an important, highly regulated process at the end of development. Senescence is characterized by breakdown of organelles and molecules, export and transport of these nutrients to other organs/parts of the organism, and finally programmed cell death of the senescing organ.The process of senescence has been intensively studied in leaves, and morphological as well as molecular changes in senescing leaves have been described. Yellowing as a consequence of chlorophyll and chloroplast degradation is the most obvious process during natural leaf senescence. In addition, gene expression changes dramatically during senescence. Some senescence-associated genes (SAG, SEN) have been reported that are induced during this process, and several of the encoded proteins function in macromolecule degradation, detoxification and defense metabolism, or signal transduction (Gepstein et al., 2003). Based on the degradation of chloroplasts and macromolecules, leaf metabolism changes from carbon assimilation to catabolism (Lim et al., 2007).The initiation and progression of senescence is regulated by endogenous as well as exogenous factors. Among the endogenous factors, the developmental status of the organ and of the whole plant (e.g. age and progress in flowering and seed production) has a great impact on the process of senescence. Different stress factors such as pathogen attack, drought, osmotic stress, heat, cold, ozone, UV light, and shading can induce or accelerate senescence (Quirino et al., 2000). Phytohormones are very important regulators that integrate information about the developmental status and the environmental factors. Cytokinins are antagonistic signals and delay senescence. Endogenous levels of cytokinins decrease during senescence, and exogenous application and transgenic approaches, enhancing endogenous levels of these compounds, lead to delayed senescence (Gan and Amasino, 1995). In contrast, the gaseous phytohormone ethylene is known to induce and accelerate senescence (John et al., 1995). There are also several indications that abscisic acid modulates senescence (van der Graaff et al., 2006). The roles of other phytohormones/signaling compounds such as auxin, salicylic acid, and jasmonates are less clear (Lim et al., 2007).Jasmonates are oxylipin signaling molecules derived from linolenic acid. The term jasmonates comprises 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives such as the methyl ester and amino acid conjugates of JA. One of the first biological activities described for these compounds was the promotion of senescence in oat (Avena sativa) leaves by methyl jasmonate (MeJa) isolated from Artemisia absinthium (Ueda and Kato, 1980). Later on, the induction of senescence-like phenotypes by exogenous application of MeJa was also found in other plant species (Ueda and Kato, 1980; Weidhase et al., 1987a; He et al., 2002). On the molecular level, this senescence-promoting effect of MeJa is accompanied by chlorophyll loss and decreases in Rubisco and photosynthesis (Weidhase et al., 1987a, 1987b). In addition, expression of some senescence-up-regulated genes is also responsive to JA; examples are SEN1, SEN4, SEN5, SAG12, SAG14, and SAG15 (Park et al., 1998; Schenk et al., 2000; He et al., 2002). Due to the results described above, jasmonates have been described for decades as compounds with senescence-promoting activities, while the function of these compounds in natural senescence in planta was critically discussed (Parthier, 1990; Sembdner and Parthier, 1993; Creelman and Mullet, 1997; Wasternack, 2007; Balbi and Devoto, 2008; Reinbothe et al., 2009). Additional indications for a role of jasmonates in regulating senescence are the transient up-regulation of expression of some enzymes involved in JA biosynthesis, such as allene oxide synthase (AOS) and OPDA reductase 3 (OPR3), and the increase in JA levels during natural senescence (He et al., 2002; van der Graaff et al., 2006). Furthermore, alterations in natural and induced senescence have been reported for some mutants with defects in the JA pathway. The mutant coi1, which is impaired in JA signaling, exhibited delayed chlorophyll loss upon dark incubation of detached leaves (Castillo and Leon, 2008). Plants with reduced expression of the 3-ketoacyl-CoA-thiolase KAT2, which is involved in β-oxidation and JA production, showed delayed yellowing during natural senescence and upon dark incubation of detached leaves (Castillo and Leon, 2008).However, there are also several reports that cast doubt on an important function of JA in senescence. For most mutants in JA biosynthesis or signaling, no differences in natural senescence are apparent (He et al., 2002; Schommer et al., 2008). In addition, mutants defective in the expression of AOS or OPR3 do not show altered senescence-like phenotypes upon dark treatment (Schommer et al., 2008; Kunz et al., 2009). It has to be taken into consideration that the knockout in these mutants has pleiotrophic effects during whole plant development. For example, the leaves of plants with reduced expression of the lipase DGL or of OPR3 are larger (Hyun et al., 2008). In addition, several knockout mutants defective in JA biosynthesis or signaling do not produce fertile flowers (Feys et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; von Malek et al., 2002). These changes in development might affect other developmental processes such as senescence.To investigate the function of jasmonates in senescence in more detail, we compared the oxylipin profile of wild-type leaves during natural senescence and upon stress induction of senescence-like phenotypes. The analysis of lipoxygenase 2 (LOX2)-RNA interference (RNAi) plants, which produce low basal levels of oxylipins but are impaired in the accumulation of OPDA and JA during senescence or in response to stress, indicates that 13-LOX products are not necessary for natural senescence or dark-induced chlorophyll loss but are involved in the response to sorbitol.     

The type IV pilin,PilA, is required for full virulence of <Emphasis Type="Italic">Francisella tularensis</Emphasis> subspecies <Emphasis Type="Italic">tularensis</Emphasis>

Background  

All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.     

Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments.To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1α,2β,4β,7β)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0^2,4]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation.     

Species-specific and leaf-age dependent effects of ultraviolet radiation on two Brassicaceae

Ultraviolet (UV) radiation affects the chemical composition of a plant. Since young leaves are of higher value due to their increased photosynthetic activity, for these a more efficient protection and thus stronger responses to a short-term exposure to natural radiation including or excluding UV-A plus UV-B radiation ("+UV" vs. "-UV") were expected than for old leaves. Nutrients and characteristic secondary metabolites of two species of Brassicaceae were analysed after two days exposure in foil-tents with different UV filtering qualities. Contents of water, carbon, nitrogen and soluble protein were found to be affected by both UV and leaf-age in Sinapis alba L. but mainly by leaf-age in Nasturtium officinale L. Glucosinolates and myrosinases, both partners of the defence system of Brassicaceae, responded highly species-specific to UV exposure. Moreover, leaf-age mainly affected total glucosinolate concentrations in S. alba, but myrosinase activities in N. officinale. The most pronounced response to UV was found in the accumulation of flavonoids which are needed to shield the leaf interior against UV. In S. alba, relative contents of quercetin flavonols increased at the expense of kaempferols in +UV exposed leaves. In N. officinale, total flavonoid quantities were 10-fold lower in -UV exposed young leaves compared to S. alba, and flavonoid accumulation was induced by UV specifically in old leaves. Hydroxycinnamic acid concentrations were not affected in both species. In total, these herbaceous species showed a highly species-specific and age-dependent plasticity in response to short-term exposure to UV which is discussed with respect to their defence strategies.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Dual effect of acid pH on purinergic P2X3 receptors depends on the histidine 206 residue

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X(3) receptors. In HEK293 cells permanently transfected with human (h) P2X(3) receptors (HEK293-hP2X(3) cells), an acidic pH shifted the concentration-response curve for alpha,beta-methylene ATP (alpha,beta-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of alpha,beta-meATP. Further, a low pH value increased the activation time constant (tau(on)) of the alpha,beta-meATP current; the fast and slow time constants of desensitization (tau(des1), tau(des2)) were at the same time also increased. Finally, acidification accelerated the recovery of P2X(3) receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X(3) receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the alpha,beta-meATP currents. The voltage sensitivity and reversal potential of the currents activated by alpha,beta-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X(3) receptor channels. It is suggested that acidification may have a dual effect on P2X(3) channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues.     

Cutting Edge: Regulatory T cells prevent efficient clearance of Mycobacterium tuberculosis

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing approximately 2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1(tm1Mom) mice (Rag1(-/-)), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-gamma- or TNF-alpha-expressing cells or general expression levels of IFN-gamma or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.