Chronic social stress induces DNA methylation changes at an evolutionary conserved intergenic region in chromosome X

Chronic stress resulting from prolonged exposure to negative life events increases the risk of mood and anxiety disorders. Although chronic stress can change gene expression relevant for behavior, molecular regulators of this change have not been fully determined. One process that could play a role is DNA methylation, an epigenetic process whereby a methyl group is added onto nucleotides, predominantly cytosine in the CpG context, and which can be induced by chronic stress. It is unknown to what extent chronic social defeat, a model of human social stress, influences DNA methylation patterns across the genome. Our study addressed this question by using a targeted-capture approach called Methyl-Seq to investigate DNA methylation patterns of the dentate gyrus at putative regulatory regions across the mouse genome from mice exposed to 14 days of social defeat. Findings were replicated in independent cohorts by bisulfite-pyrosequencing. Two differentially methylated regions (DMRs) were identified. One DMR was located at intron 9 of Drosha, and it showed reduced methylation in stressed mice. This observation replicated in one of two independent cohorts. A second DMR was identified at an intergenic region of chromosome X, and methylation in this region was increased in stressed mice. This methylation difference replicated in two independent cohorts and in Major Depressive Disorder (MDD) postmortem brains. These results highlight a region not previously known to be differentially methylated by chronic social defeat stress and which may be involved in MDD.     

Continuous micropropagation of juvenile larch from different species via adventitious bud formation

A continuous propagation of juvenile larch in vitro was based on adventitious bud formation and different cytokinin combinations were tested concerning their effectiveness to induce elongation of adventitious buds. Zeatin (1.5 mg dm-3) combined with kinetin (0.15 mg dm-3) was found to be the best. Development and elongation of buds was achieved on a modified LP-medium. Using this system it was possible to propagate different larchspecies (Larix decidua, L. gmelinii and L. sukaczewii f. multiramosus) continuously. Shoots were successfully rooted and transferred to the soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sucrose Accumulation in the Sugarcane Stem Is Regulated by the Difference between the Activities of Soluble Acid Invertase and Sucrose Phosphate Synthase

To assess the relative importance of morphological and biochemical factors in the regulation of sucrose (Suc) accumulation in the sugarcane (Saccharum spp. hybrids) stem, we investigated morphological and biochemical correlates of Suc accumulation among parents and progeny of a family segregating for differences. In contrast to the parents, no relationship was observed between morphology and the level of Suc accumulation among the progeny. The level and timing of Suc accumulation in the whole stalk and within individual internodes was correlated with the down-regulation of soluble acid invertase (SAI) activity. High SAI activity prevented most, but not all, Suc accumulation. There was a critical threshold of SAI activity above which high concentrations of Suc did not accumulate. This low level of SAI activity was always exceeded in the internodes of the lower-Suc-storing genotypes. However, low activity of SAI was not sufficient by itself to account for the Suc accumulation in the higher-Suc-storing genotypes. Major differences in Suc accumulation among the population were attributed to the difference between activities of SAI and Suc phosphate synthase, provided SAI is below the critical threshold concentration. This result is not unexpected, since the pathway of Suc transport for storage involves Suc hydrolysis and resynthesis.     

A confirmation of the proposed model for the hexose uptake system ofChlorella vulgaris. Anaerobic studies in the light and in the dark

Summary Energy for accumulation of sugar analogues inChlorella vulgaris can be supplied by respiration and by light. Under anaerobic conditions a quantum efficiency of close to one has been determined for light-dependent uptake of 6-deoxyglucose at 712 nm. Although the rate of uptake under anaerobic conditions in the dark is less than 15% of that in the light, the steady-state plateau of accumulation does not change when the light is turned off. This is explained by the observation that efflux of sugar is inhibited by lack of energy to the same extent as influx. This agrees with a model proposed earlier (Komor, Haass & Tanner,Biochim. Biophys. Acta 266:649, 1972). Under aerobic conditions during the steady-state plateau in the dark, the rate of influx of 6-deoxyglucose is twice that of initial influx (positive transmembrance effect). By anaerobiosis the initial influx is almost completely inhibited, whereas the steady-state influx is affected only to 50%. This points to an energyless homoexchange. However, this flux is completely inhibited by uncoupling agents. To explain these results, the existence of an energygenerating efflux has been assumed, which is affected by uncouplers. The efficiency of energy production by efflux and the re-use of this energy for influx is inversely related to the amount of energy supplied by other energy-generating processes in the cells; under anaerobic conditions in the dark, 90% of the efflux energy seems to be available for influx.     

Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.     

Ochratoxin A in human blood serum – retrospective long-term data

In a long-term study (1990–1997) on ochratoxin A (OTA) in human blood serum, 102 serum samples from 36 persons of the Munich Institute for Hygiene and Technology of Food of Animal Origin were analysed by enzyme immunoassay (EIA), and by high performance liquid chromatography (HPLC) for control. Detection limits were at 50 pg/ml (EIA) and 50–70 pg/ml (HPLC), recoveries were 80–120% (EIA) and 30–60% (LC). OTA was detected in 98% (EIA, 368 ± 217 pg/ml) and 93% (HPLC, 271 ± 170 pg/ml) of samples (maximum 1,290 pg/ml). Using published conversion factors for serum/intake estimates (1.34 or 1.97), the mean daily OTA intake of these 36 persons was 493–725 pg/kg bw. Long-term individual mean OTA levels of nine persons ranged from 162 ± 80 pg/ml to 549 ± 172 pg/ml. Our data were compared with published OTA serum levels (1985–2008) for apparently healthy persons from a total of 30 countries. On a worldwide basis, the mean of means for OTA in human serum was estimated to be 700 pg/ml, corresponding to a mean daily OTA intake of 940–1380 pg/kg bw. This level, which was relatively stable over the last decades, is well below published tolerable daily intake values (14,000–18,000 pg/kg bw).     

Mapping an invasive bryophyte species using hyperspectral remote sensing data

Reliable distribution maps are crucial for the management of invasive plant species. An alternative to traditional field surveys is the use of remote sensing data, which allows coverage of large areas. However, most remote sensing studies on invasive plant species focus on mapping large stands of easily detectable study species. In this study, we used hyperspectral remote sensing data in combination with field data to derive a distribution map of an invasive bryophyte species, Campylopus introflexus, on the island of Sylt in Northern Germany. We collected plant cover data on 57 plots to calibrate the model and presence/absence data of C. introflexus on another 150 plots for independent validation. We simultaneously acquired airborne hyperspectral (APEX) images during summer 2014, providing 285 spectral bands. We used a Maxent modelling approach to map the distribution of C. introflexus. Although C. introflexus is a small and inconspicuous species, we were able to map its distribution with an overall accuracy of 75 %. Reducing the sampling effort from 57 to 7 plots, our models performed fairly well until sampling effort dropped below 12 plots. The model predicts that C. introflexus is present in about one quarter of the pixels in our study area. The highest percentage of C. introflexus is predicted in the dune grassland. Our findings suggest that hyperspectral remote sensing data have the potential to provide reliable information about the degree of bryophyte invasion, and thus provide an alternative to traditional field mapping approaches over large areas.     

Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis

Prosthetic lipoyl groups are required for the function of several essential multienzyme complexes, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and the glycine cleavage system (glycine decarboxylase [GDC]). How these proteins are lipoylated has been extensively studied in prokaryotes and yeast (Saccharomyces cerevisiae), but little is known for plants. We earlier reported that mitochondrial fatty acid synthesis by ketoacyl-acyl carrier protein synthase is not vital for protein lipoylation in Arabidopsis (Arabidopsis thaliana) and does not play a significant role in roots. Here, we identify Arabidopsis lipoate-protein ligase (AtLPLA) as an essential mitochondrial enzyme that uses octanoyl-nucleoside monophosphate and possibly other donor substrates for the octanoylation of mitochondrial PDH-E2 and GDC H-protein; it shows no reactivity with bacterial and possibly plant KGDH-E2. The octanoate-activating enzyme is unknown, but we assume that it uses octanoyl moieties provided by mitochondrial β-oxidation. AtLPLA is essential for the octanoylation of PDH-E2, whereas GDC H-protein can optionally also be octanoylated by octanoyltransferase (LIP2) using octanoyl chains provided by mitochondrial ketoacyl-acyl carrier protein synthase to meet the high lipoate requirement of leaf mesophyll mitochondria. Similar to protein lipoylation in yeast, LIP2 likely also transfers octanoyl groups attached to the H-protein to KGDH-E2 but not to PDH-E2, which is exclusively octanoylated by LPLA. We suggest that LPLA and LIP2 together provide a basal protein lipoylation network to plants that is similar to that in other eukaryotes.Lipoic acid (LA; 6,8-dithiooctanoic acid) prosthetic groups are essential for the catalytic activity of four important multienzyme complexes in plants and other organisms: pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), branched-chain α-ketoacid dehydrogenase (BCDH), and the Gly cleavage system (glycine decarboxylase [GDC]; Perham, 2000; Douce et al., 2001; Mooney et al., 2002). In all these multienzyme complexes, LA is covalently attached to the ε-amino group of a particular lysyl residue of the respective protein subunit. Lipoylated E2 subunits of PDH, KGDH, and BCDH are dihydrolipoyl acyltransferases that interact with E1 and E3 subunits to pass acyl intermediates to CoA (Mooney et al., 2002). By contrast, the lipoylated H-protein of GDC acts as a cosubstrate of three other GDC proteins and has no enzymatic activity itself (Douce et al., 2001). In the course of their respective reaction cycles, LA becomes reduced to dihydrolipoic acid. Most of these enzymes are confined to the mitochondrion. As the only exception, PDH is also present in plastids, where it provides acetyl-CoA for fatty acid biosynthesis (Ohlrogge et al., 1979; Lernmark and Gardeström, 1994; Lin et al., 2003).Mitochondria and plastids each have their own route of de novo LA synthesis, both of which start with the synthesis of protein-bound octanoyl chains (Shimakata and Stumpf, 1982; Ohlrogge and Browse, 1995; Wada et al., 1997; Gueguen et al., 2000; Yasuno et al., 2004). These octanoyl moieties are passed on by organelle-specific octanoyltransferases (Wada et al., 2001a, 2001b) to the respective target apoproteins where lipoyl synthase (LIP1) inserts two sulfur atoms to finally produce functional lipoyl groups (Yasuno and Wada, 1998, 2002; Zhao et al., 2003). A similar pathway has been identified in mammalian mitochondria (Morikawa et al., 2001; Witkowski et al., 2007). In quantitative terms, leaf mesophyll mitochondria have an extraordinarily high requirement for lipoate, because they contain very large amounts of GDC to catalyze the photorespiratory Gly-to-Ser conversion (Bauwe et al., 2010). For this reason, leaf mesophyll mitochondria are the major site of LA synthesis in plants (Wada et al., 1997).It was thought that the octanoyl chains provided by mitochondrial β-ketoacyl-acyl carrier protein synthase (mtKAS) represent the solitary source for protein lipoylation in plant mitochondria (Yasuno et al., 2004). As we reported earlier, however, leaves of mtKAS-deficient knockout mutants show considerable lipoylation of mitochondrial PDH-E2 and KGDH-E2 subunits and some residual lipoylation of GDC H-protein; roots are not at all impaired. Accordingly, the phenotype of such mutants can be fully cured in the low-photorespiratory condition of elevated CO₂ (Ewald et al., 2007). These observations indicated that plant mitochondria, in addition to the mtKAS-LIP2-LIP1 route of protein lipoylation, can resort to an alternative pathway. This would not be uncommon. In Escherichia coli, for example, a salvage pathway utilizes free octanoate or LA in an ATP-dependent two-step reaction catalyzed by the bifunctional enzyme lipoate-protein ligase A (LPLA; Morris et al., 1995). Archaea (Christensen and Cronan, 2009; Posner et al., 2009) and vertebrates (Tsunoda and Yasunobu, 1967) require two separate enzymes to first activate octanoate or LA to lipoyl-nucleoside monophosphate (NMP) and then, in a second step, to convey the activated lipoyl group to the respective target proteins. The lipoate-activating enzyme (LAE) of mammals was identified as a refunctioned medium-chain acyl-CoA synthetase that utilizes GTP to produce lipoyl-GMP (Fujiwara et al., 2001). LIP3 from yeast (Saccharomyces cerevisiae) can use octanoyl-CoA to octanoylate apoE2 proteins (Hermes and Cronan, 2013), whereas octanoyl groups from fatty acid biosynthesis are first attached to H-protein and then passed on to apoE2 proteins (Schonauer et al., 2009).The physiological significance of lipoyl-protein ligases in plants is not exactly known. Such enzymes do not operate in plastids (Ewald et al., 2014) but could be present in mitochondria. A single-gene-encoded LPLA with predicted mitochondrial localization has been identified in rice (Oryza sativa; Kang et al., 2007). Complementation studies with the lipoylation-deficient E. coli mutant TM137 (Morris et al., 1995) suggested that OsLPLA belongs to the bifunctional type of LPLAs. We report the identification of the homologous enzyme in Arabidopsis (Arabidopsis thaliana), provide evidence for its mitochondrial location, and show that Arabidopsis LPLA requires a separate enzyme for octanoate/lipoate activation. We also examine the interplay between LPLA, LIP2, and the mtKAS route of protein lipoylation and suggest a model for protein lipoylation in plant mitochondria.     

Selective cytotoxicity of rhodium metalloinsertors in mismatch repair-deficient cells

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anticancer agents.