Reversal of Abscisic Acid-Induced Stomatal Closure by trans-Cinnamic and p-Coumaric Acid

Abscisic acid (ABA)-induced increase in stomatal diffusive resistance (SDR) in excised leaves of bean (Phaseolus vulgaris L. cv Pencil Pod) and maize (Zea mays L. cv Golden Bantam) is inhibited by low concentrations of trans-cinnamic acid (TCA) (1 micromolar) and p-coumaric acid (PCA) (10 micromolar) when given together with ABA (10 micromolar) in the transpiration stream through the cut end of the petiole or leaf blade. A concentration effect is observed both in the ABA action and its reversal by phenolic acids. Leaves having attained a high diffusive resistance in ABA solution recover rapidly when transferred to water. ABA (10 micromolar) induced closure of the stomata in onion, Allium cepa L. and Vicia faba epidermal peels. This is associated with loss of K⁺ from guard cells. In the presence of TCA (10 micromolar) and PCA (10 micromolar) K⁺ is retained in the guard cells with open stomata. The dark closure of stomata is also inhibited by TCA and PCA. It is suggested that these phenolic acids may inhibit the ABA effect by competing with or acting on some ABA-specific site, probably located on the plasma membrane, regulating flux of K⁺ ions. A weak association of ABA with the plasma membrane is envisaged because of the rapid recovery obtained upon transferral of the leaves to water.     

Long‐term seed bank dynamics in a temperate forest under conversion from coppice‐with‐standards to high forest management

Questions: How do changes in forest management, i.e. in disturbance type and frequency, influence species diversity, abundance and composition of the seed bank? How does the relationship between seed bank and vegetation change? What are the implications for seed bank dynamics? Location: An ancient Quercus petraea — Carpinus betulus forest in conversion from coppice‐with‐standards to regular Quercus high forest near Montargis, France. Methods: Seed bank and vegetation were sampled in six replicated stand types, forming a chronosequence along the conversion pathway. The stand types represented mid‐successional stages of stands in transition from coppice‐with‐standards (to high forest (16 plots) and early‐ and mid‐successional high forest stands (32 plots). Results: Seed bank density and species richness decreased with time since last disturbance. Adjusting for seed density effects obscured species richness differences between stand types, but species of later seres were nested subsets of earlier seres, implying concomitant shifts in species richness and composition with time since disturbance. Later seres were characterized by species with low seed weight and high seed longevity. Seed banks of early seres were more similar to vegetation than to later seres. Conclusions: Abandonment of the coppice‐with‐standards regime altered the seed bank characteristics, as well as its relationship with vegetation. Longer management cycles under high forest yield impoverished seed banks. For their persistence, seed bank species will increasingly rely on management of permanently open areas in the forest landscape. Thus, revegetation at the beginning of new high‐forest cycles may increasingly depend on inflow from seed sources.     

M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model

The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4⁺ and particularly CD8⁺ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG.     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

A novel approach for extraction of PCR-compatible DNA from activated sludge samples collected from different biological effluent treatment plants

This paper describes a method that facilitates the extraction of PCR-compatible DNA from different activated sludge samples. The approach involves a novel preprocessing step in DNA extraction, which removes potential PCR inhibitors. The sludge was washed with different ratios of acetone and petroleum ether after pretreatment with 0.01% Tween-20 at 50 degrees C. It was observed that an initial washing step with 50 mM Tris-HCl, pH 9.0, before the detergent-solvent step, improved the quality of the extracted DNA. The extraction protocol resulted in amplifiable amounts of DNA when 10 mg of a sludge sample was used, even in the presence of phenol as a sludge contaminant. The usefulness of the extracted template was demonstrated by carrying out different PCR reactions. The random amplified polymorphic DNA (RAPD) patterns demonstrated the diversity of sludge samples.     

Steroid sulfatase inhibitors: promising new tools for breast cancer therapy?

Inhibition of aromatase is currently well-established as the major treatment option of hormone-dependent breast cancer in postmenopausal women. However, despite the effects of aromatase inhibitors in both early and metastatic breast cancer, endocrine resistance may cause relapses of the disease and progression of metastasis. Thus, driven by the success of manipulating the steroidogenic enzyme aromatase, several alternative enzymes involved in steroid synthesis and metabolism have recently been investigated as possible drug targets. One of the most promising targets is the steroid sulfatase (STS) which converts steroid sulfates like estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) to estrone (E1) and dehydroepiandrosterone (DHEA), respectively. Estrone and DHEA may thereafter be used for the synthesis of more potent estrogens and androgens that may eventually fuel hormone-sensitive breast cancer cells. The present review summarizes the biology behind steroid sulfatase and its inhibition, the currently available information derived from basic and early clinical trials in breast cancer patients, as well as ongoing research. Article from the Special Issue on Targeted Inhibitors.     

Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes. Sequences of NtCAD1-1 and NtCAD1-7 were deposited in GenBank under accession numbers AY911854 and AY911855, respectively.     

Home on the range: spatial ecology of loggerhead turtles in Atlantic waters of the USA

Aim Although satellite tracking has yielded much information regarding the migrations and habitat use of threatened marine species, relatively little has been published about the environmental niche for loggerhead sea turtles Caretta caretta in north‐west Atlantic waters. Location North Carolina, South Carolina and Georgia, USA. Methods We tracked 68 adult female turtles between 1998 and 2008, one of the largest sample sizes to date, for 372.2 ± 210.4 days (mean ± SD). Results We identified two strategies: (1) ‘seasonal’ migrations between summer and winter coastal areas (n = 47), although some turtles made oceanic excursions (n = 4) and (2) occupation of more southerly ‘year‐round’ ranges (n = 18). Seasonal turtles occupied summer home ranges of 645.1 km² (median, n = 42; using α‐hulls) predominantly north of 35 ° latitude and winter home ranges of 339.0 km² (n = 24) in a relatively small area on the narrow shelf off North Carolina. We tracked some of these turtles through successive summer (n = 8) and winter (n = 3) seasons, showing inter‐annual home range repeatability to within 14.5 km of summer areas and 10.3 km of winter areas. For year‐round turtles, home ranges were 1889.9 km². Turtles should be tracked for at least 80 days to reliably estimate the home range size in seasonal habitats. The equivalent minimum duration for ‘year‐round’ turtles is more complex to derive. We define an environmental envelope of the distribution of North American loggerhead turtles: warm waters (between 18.2 and 29.2 °C) on the coastal shelf (in depths of 3.0–89.0 m). Main conclusions Our findings show that adult female loggerhead turtles show predictable, repeatable home range behaviour and do not generally leave waters of the USA, nor the continental shelf (< 200m depth). These data offer insights for future marine management, particularly if they were combined with those from the other management units in the USA.