The Combined Dexamethasone/CRH Test (DEX/CRH Test) and Prediction of Acute Treatment Response in Major Depression

Background

In this study the predictive value of the combined dexamethasone/CRH test (DEX/CRH test) for acute antidepressant response was investigated.

Methodology/Principal Findings

In 114 depressed inpatients suffering from unipolar or bipolar depression (sample 1) the DEX/CRH test was performed at admission and shortly before discharge. During their stay in the hospital patients received different antidepressant treatment regimens. At admission, the rate of nonsuppression (basal cortisol levels >75.3 nmol/l) was 24.6% and was not related to the later therapeutic response. Moreover, 45 out of 114 (39.5%) patients showed an enhancement of HPA axis function at discharge in spite of clinical improvement. In a second sample, 40 depressed patients were treated either with reboxetine or mirtazapine for 5 weeks. The DEX/CRH test was performed before, after 1 week, and after 5 weeks of pharmacotherapy. Attenuation of HPA axis activity after 1 week was associated with a more pronounced alleviation of depressive symptoms after 5-week mirtazapine treatment, whereas downregulation of HPA system activity after 5 weeks was related to clinical response to reboxetine. However, early improvement of HPA axis dysregulation was not necessarily followed by a beneficial treatment outcome.

Conclusions/Significance

Taken together, performance of a single DEX/CRH test does not predict the therapeutic response. The best predictor for response seems to be an early attenuation of HPA axis activity within 1 or 2 weeks. However, early improvement of HPA system dysfunction is not a sufficient condition for a favourable response. Since a substantial part of depressive patients display a persistence of HPA axis hyperactivity at discharge, downregulation of HPA system function is not a necessary condition for acute clinical improvement either. Our data underline the importance of HPA axis dysregulation for treatment outcome in major depression, although restoration of HPA system dysfunction seems to be neither a necessary nor a sufficient determinant for acute treatment response.     

RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background

Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/Principal Findings

Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/Significance

Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.     

EDDIE fusion proteins: Triggering autoproteolytic cleavage

Heterologous proteins are often poorly expressed in Escherichia coli and especially small peptides are prone to degradation. N^pro autoprotease fusion proteins, deposited as inclusion bodies in E. coli, are a versatile tool for peptide and protein overexpression and generate an authentic N terminus at the target molecule. Autoproteolytic cleavage and subsequent release of the fusion partner are initiated upon refolding. Fusion proteins with the N^pro mutant EDDIE follow a monomolecular reaction. The reaction rate was only dependent on chaotrope concentration, decreasing exponentially by a factor of 1.2–1.5 for urea and by a factor of 2.1–5.3 for GuHCl. The first amino acid of the target peptide had a major impact on the reaction rate studying a set of model peptides. Reaction rates were in the range of 2.2 × 10^?4 to 7.3 × 10^?5 s^?1 and could be increased up to fivefold by exchanging the first amino acid of the target peptide. A panel of biophysical methods was used to assess EDDIE secondary and tertiary structure. Immediate formation of secondary structure and slight increase in β-sheet content of approximately 5% over the course of the cleavage reaction was observed and interpreted as aggregation. Aggregation and cleavage occurred simultaneously. EDDIE has a relatively loose structure with the cleavage site exhibiting the lowest solvent exposure. We hypothesize that this is the mechanism for establishing a spatial proximity between cleavage site and the catalytic centre of the autoprotease. Fluorescence measurements revealed that further structural changes did not occur after the initial hydrophobic collapse. Thus, the overall reaction is predominantly controlled by cleavage kinetics and refolding kinetics does not play a major role.     

Reversible Phosphorylation as a Molecular Switch to Regulate Plasma Membrane Targeting of Acylated SH4 Domain Proteins

Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes. Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated down-regulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.     

More than anticipated - production of antibiotics and other secondary metabolites by Bacillus amyloliquefaciens FZB42

The genome of environmental Bacillus amyloliquefaciens FZB42 harbors numerous gene clusters involved in synthesis of antifungal and antibacterial acting secondary metabolites. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137 kb, direct non-ribosomal synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron siderophore bacillibactin. Bacillomycin and fengycin were shown to act against phytopathogenic fungi in a synergistic manner. Three gene clusters, mln, bae, and dif, with a total length of 199 kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. Both, non-ribosomal synthesis of cyclic lipopeptides and synthesis of polyketides are dependent on the presence of a functional sfp gene product, 4'-phosphopantetheinyl transferase, as evidenced by knockout mutation of the sfp gene resulting in complete absence of all those eight compounds. In addition, here we present evidence that a gene cluster encoding enzymes involved in synthesis and export of the antibacterial acting dipeptide bacilysin is also functional in FZB42. In summary, environmental FZB42 devoted about 340 kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites useful to cope with other competing microorganisms present in the plant rhizosphere.     

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning,Sugar Signaling,and Seed Yield in Arabidopsis

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyll_ab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO₂ release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development.Sugars fulfill an extraordinarily wide range of functions in plants as well as in other organisms. They serve as valuable energy resources that are easy to store and remobilize. Sugars are required for the synthesis of cell walls and carbohydrate polymers. They are also necessary for starch accumulation and serve as precursors for a range of primary and secondary plant intermediates. From a chemical point of view, sugars represent a large class of metabolites. Among the prominent members in higher plants are the monosaccharides Glc and Fru and the disaccharide Suc (ap Rees, 1994).In contrast to heterotrophic organisms, plants are able to synthesize sugars de novo and to degrade them via oxidative or fermentative metabolism (Heldt, 2005). Net sugar accumulation in plants takes place during the day, whereas net degradation of stored carbohydrate reserves takes place the following night. In higher plants, autotrophic and heterotrophic organs appear to be interconnected by phloem for long-distance transport of sugars (Ruiz-Medrano et al., 2001). Accordingly, sugars must be transported within cells, between cells, and between plant organs. Given these factors, along with the outstanding importance of sugars, it is not surprising that plants sense intracellular sugar availability and use this information to coordinate the expression of many genes (Koch, 1996; Moore et al., 2003).In Arabidopsis (Arabidopsis thaliana), about 60 genes code for putative monosaccharide transport proteins and about 10 genes encode predicted disaccharide carriers (Lalonde et al., 2004). Transport of neutral sugars has been monitored across the plasma membrane, the chloroplast envelope, and the vacuolar membrane (Weber et al., 2000; Niittylä et al., 2004; Martinoia et al., 2007). So far, all sugar carriers residing in the plant plasma membrane have been characterized to catalyze proton-coupled sugar movement (Sauer, 1992; Büttner and Sauer, 2000; Carpaneto et al., 2005). In contrast, both facilitated diffusion and proton-driven antiport mechanisms have been described for monosaccharide and Suc transport across the vacuolar membrane (Thom and Komor, 1984; Daie and Wilusz, 1987; Martinoia et al., 1987; Shiratake et al., 1997; Neuhaus, 2007).In plants, vacuoles fulfill critical functions in the long-term and temporary storage of sugars, sugar alcohols, and other primary metabolites such as carboxylates and amino acids (Dietz et al., 1990; Rentsch and Martinoia, 1991; Martinoia and Rentsch, 1992; Emmerlich et al., 2003). Recently, the first solute carriers responsible for vacuolar Suc and inositol transport have been identified (Endler et al., 2006; Schneider et al., 2008). In addition, TMT (for tonoplast monosaccharide transporter) and VGT (for vacuolar Glc transporter) were the first vacuolar carrier proteins proven to have transport capacity for both Glc and Fru (Wormit et al., 2006; Aluri and Büttner, 2007).TMT exists in three isoforms in Arabidopsis (TMT1–TMT3), and orthologs have been found in other plant species like grapevine (Vitis vinifera), barley (Hordeum vulgare), and rice (Oryza sativa; Wormit et al., 2006). In Arabidopsis, the genes TMT1 and TMT2 are expressed in various tissues, whereas TMT3 is hardly expressed throughout the entire plant life cycle (Wormit et al., 2006). Interestingly, TMT1 and TMT2 are induced by Glc, salt, drought, and cold stress (Wormit et al., 2006), and vacuoles isolated from a TMT1 loss-of-function (T-DNA) Arabidopsis mutant showed reduced Glc import capacity in comparison with corresponding wild-type organelles (Wormit et al., 2006). Moreover, after transfer into the cold, these mutant leaves showed impaired ability to accumulate Glc and Fru, underscoring the in vivo function of TMT under selected conditions (Wormit et al., 2006).However, it is unknown to what extent overexpression of a vacuolar sugar carrier affects subcellular sugar allocation in Arabidopsis. In addition, whether increased vacuolar sugar transport influences sugar signaling, plant development, or organ properties has not been determined. Thus, it is unknown how important controlled activity of vacuolar monosaccharide transport is to plant development or physiological properties. To reveal whether TMT activity affects these processes, we created TMT1-overexpressing Arabidopsis lines and analyzed their physiological and molecular feedbacks.     

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with ¹³C and ¹⁵N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of ¹³C and ¹⁵N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of ¹³C and ¹⁵N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with ¹³C and ¹⁵N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.     

Antimony uptake and toxicity in sunflower and maize growing in SbIII and SbV contaminated soil

Using pot experiments, we investigated the uptake of antimony (Sb) by sunflower (Helianthus annuus L. cv. Iregi), and maize (Zea mays L. cv. Magister) in two different soils, a potting mix and an agricultural soil. In one treatment Sb was added to the experimental soils as KSb(OH)₆ (“Sb^V-treatment”) and in the other as Sb₂O₃ (“Sb^III-treatment”). Soluble soil Sb concentrations were linearly related to the applied Sb rates, ranging from 0.02 (controls) to 175 mg L^?1 soil solution. Accumulation of Sb tended to be slightly higher in the Sb^V treatment in sunflower, while no difference in Sb uptake between the two Sb treatments was found in maize. The half maximal effective concentration (EC₅₀) values derived from the dose-response curves were higher for the Sb^V than for the Sb^III treatment when they were related to soluble soil Sb concentrations, but differences became insignificant when they were related to shoot Sb concentrations. Maize was substantially more sensitive to Sb toxicity than sunflower, indicating physiological differences in Sb tolerance between the two plant species. Our results show that on soils with high Sb contamination, as often found in shooting ranges, plants may suffer from Sb toxicity.