Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene

The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant, particularly at military sites and former manufacturing facilities, presents various health and environmental concerns. Due to the chemically resistant structure of TNT, it has proven to be highly recalcitrant to biodegradation in the environment. Here, we demonstrate the importance of two glutathione transferases (GSTs), GST-U24 and GST-U25, from Arabidopsis (Arabidopsis thaliana) that are specifically up-regulated in response to TNT exposure. To assess the role of GST-U24 and GST-U25, we purified and characterized recombinant forms of both enzymes and demonstrated the formation of three TNT glutathionyl products. Importantly, GST-U25 catalyzed the denitration of TNT to form 2-glutathionyl-4,6-dinitrotoluene, a product that is likely to be more amenable to subsequent biodegradation in the environment. Despite the presence of this biochemical detoxification pathway in plants, physiological concentrations of GST-U24 and GST-U25 result in only a limited innate ability to cope with the levels of TNT found at contaminated sites. We demonstrate that Arabidopsis plants overexpressing GST-U24 and GST-U25 exhibit significantly enhanced ability to withstand and detoxify TNT, properties that could be applied for in planta detoxification of TNT in the field. The overexpressing lines removed significantly more TNT from soil and exhibited a corresponding reduction in glutathione levels when compared with wild-type plants. However, in the absence of TNT, overexpression of these GSTs reduces root and shoot biomass, and although glutathione levels are not affected, this effect has implications for xenobiotic detoxification.The containment and cleanup of environmental pollutants is increasingly both a legal requirement and a responsible action in many developed countries. The most commonly used explosives in military weapons are 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and their continual use, along with production and decommissioning, are progressively contaminating millions of hectares of military land (Rylott and Bruce, 2009). Bioremediation of TNT is particularly challenging, as the electron-withdrawing properties of the three nitro groups render the aromatic ring particularly resistant to oxidative attack and ring cleavage by microbial oxygenases, which in the environment are normally central to the biodegradation of aromatic compounds (Qasim et al., 2007). In the United States, the Environmental Protection Agency and the military are addressing methods by which toxic TNT and RDX can be contained and detoxified on active military training ranges. One way this problem might be tackled is through the use of plants that are adapted to detoxify these compounds. This could be achieved either by traditional breeding programs or by genetic modification, as has been demonstrated previously for both RDX and TNT (Hannink et al., 2001; Rylott et al., 2006; Jackson et al., 2007).In the majority of species tested so far (tobacco [Nicotiana tabacum], bean [Phaseolus vulgaris], wheat [Triticum aestivum], poplar [Populus spp.], and switchgrass [Panicum virgatum]), with the exception of some conifer trees (Schoenmuth and Pestemer, 2004), TNT is located almost entirely in the roots (Sens et al., 1998, 1999; Hannink et al., 2007; Van Dillewijn et al., 2008; Brentner et al., 2010). Endogenous metabolism of TNT by plants has been characterized (Rylott and Bruce, 2009; Rylott et al., 2011b), with recent research focusing on the model plant species Arabidopsis (Arabidopsis thaliana; Hannink et al., 2001; Van Dillewijn et al., 2008; Rylott et al., 2011a). First, TNT is transformed by nitroreductases to hydroxylamino dinitrotoluenes (HADNTs), with a varying portion further reduced to amino dinitrotoluenes (ADNTs). In Arabidopsis, oxophytodienoate reductases are known to catalyze these steps (Beynon et al., 2009). Plants engineered to express bacterial nitroreductases, which also perform this transformation step, have increased TNT transformation activity and show dramatically enhanced resistance to TNT (Hannink et al., 2001; Rylott et al., 2011a). The additional functionality of HADNTs and ADNTs permits their subsequent conjugation to amino acids, organic acids, and sugars (Bhadra et al., 1999, 2001), and conjugation of HADNT and ADNT isomers to Glc by Arabidopsis glucosyltransferases has been characterized (Gandia-Herrero et al., 2008), with research suggesting that these conjugates are subsequently sequestered within the cell walls (Rylott and Bruce, 2009).Glutathione transferases (GSTs) are a multigene family of proteins known to conjugate glutathione to electrophilic molecules and, in plants, are involved in the detoxification of herbicide xenobiotics (Cummins et al., 2011). Since GSTs have evolved the ability to catalyze glutathione-linked reactions with thousands of different chemical structures, it has been hypothesized that GSTs should play a central role, alongside glucosyltransferases, in the detoxification of TNT (Mezzari et al., 2005; Brentner et al., 2008). Gene expression studies in poplar (Tanaka et al., 2007; Brentner et al., 2008) and Arabidopsis (Ekman et al., 2003; Mezzari et al., 2005; Gandia-Herrero et al., 2008) have identified GSTs up-regulated in response to TNT; however, to date, the biochemical response of GSTs toward TNT has not been investigated. The overexpression of plant GSTs has been shown to increase resistance to a range of stresses, with some τ class GSTs shown to detoxify herbicides via a conjugation activity (Dixon and Edwards, 2010; Cummins et al., 2011). Many Arabidopsis GSTs (in common with some mammalian and other plant GSTs) exhibit a glutathione-dependent peroxide (GPOX) activity (Dixon and Edwards, 2010), catalyzing the reduction of lipid hydroperoxides to the respective monohydroxyalcohols, an activity that confers tolerance to a number of oxidative stresses (Dixon et al., 1998; Dixon and Edwards, 2010). Here, we expressed, purified, and characterized TNT-responsive Arabidopsis GSTs and investigated their contribution toward the detoxification of TNT in Arabidopsis.     

Value of rapid aetiological diagnosis in optimization of antimicrobial treatment in bacterial community acquired pneumonia

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs. 160.11 mg/L, p = 0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP.     

Oral glycine administration attenuates diabetic complications in streptozotocin-induced diabetic rats

Diabetes mellitus is a disease characterized by impaired glucose metabolism that leads to retinopathy, brain micro-infarcts and other complications. We have previously shown that oral glycine administration to diabetic rats inhibits non-enzymatic glycation of hemoglobin and diminishes renal damage. In this work, we evaluated the capacity of the amino acid glycine (1% w/v, 130 mM) to attenuate diabetic complications in streptozotocin (STZ)-induced diabetic Wistar rats and compared them with non-treated or taurine-treated (0.5% w/v, 40 mM) diabetic rats. Glycine-treated diabetic rats showed an important diminution in the percentage of animals with opacity in lens and microaneurysms in the eyes. Interestingly, there was a diminished expression of O-acetyl sialic acid in brain vessels compared with untreated diabetic rats (P<0.05). Additionally, peripheral blood mononuclear cells isolated from glycine-treated diabetic rats showed a better proliferative response to PHA or ConA than those obtained from non-treated diabetic rats (P<0.05). Glycine-treated rats had a less intense corporal weight loss in comparison with non-treated animals. Our results suggest that administration of glycine attenuates the diabetic complications in the STZ-induced diabetic rat model, probably due to inhibition of the non-enzymatic glycation process.     

Matrigel-induced acinar differentiation is followed by apoptosis in HSG cells

It has been shown that a human salivary gland cell line (HSG) is capable of differentiation into gland-like structures, though little is known of how morphological features are formed or controlled. Here we investigated the changes in cell proliferation and apoptosis upon terminal differentiation of HSG cells in Matrigel, an extracellular matrix derivative. Changes in the expression of survivin, a prominent anti-apoptotic factor, and caspase-3, a key apoptotic factor were also measured. In order to better understand the involvement of key signal transduction pathways in this system we pharmacologically blocked the activity of tyrosine kinase, nuclear factor kappa B(NF kappa B), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and matrix metalloproteases (MMP). Results of these studies demonstrate that cytodifferentiation of HSG cells to an acinar phenotype is accompanied first by a decrease of cell proliferation and then by a massive programmed cell death, affected by multiple signal transduction pathways. Thus, Matrigel alone is insufficient for the full maturation and long term survival of the newly formed acini: the presence of other factors is necessary to complete the acinar differentiation of HSG cells.     

Quality of different aphids as hosts of the parasitoid Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae, Aphidiinae)

Lysiphlebus testaceipes (Cresson) has a broad aphid host range; however the quality of these preys may interfere in its biological feature. This study aimed to evaluate the quality of three Macrosiphini, Brevicoryne brassicae (L.), Lipaphis erysimi (Kaltenbach) and Myzus persicae (Sulzer), and three Aphidini Schizaphis graminum (Rondani) Rhopalosiphum maidis (Fitch) and Aphis gossypii Glover as hosts to L. testaceipes and to determine the relation possible of host preference, of size and quality of the host. The tests were carried out in climatic chamber at 25 +/- 1 degrees C, RH 70 +/- 10% and 12h photophase. The parasitoid did not oviposite in B. brassicae and L. erysimi, while the other species were nutritionally suitable to the parasitoid. L. testaceipes showed preference for aphids from tribe Aphidini and these hosts presented better quality to the parasitoid when compared to Macrosiphini. Interactions among size, preference and quality between the Aphidini were found. L testaceipes showed preference (parasitism rate 76.7%) for R. maidis, the bigger host (hind tibia with 0.281 mm). This host provided bigger size (hind tibia with 0.49 mm) and higher emergence rate (95.6%) to the parasitoid when compared to A. gossypii (parasitism rate of 55.7%). Also the smaller host A. gossypii (0.266 mm) provided smaller size hind tibia (0.45 mm) and higher mortality of the parasitoid (emergence rate 72.1%). However, the development time was shorter and the longevity was higher in A. gossypii (6.3 and 5.4 days, respectively) when compared to the host R. maidis (6.7 and 3.8 days, respectively), and not been related to host size.     

Bone Disease in Newly Diagnosed Lupus Nephritis Patients

Introduction

Bone loss in Lupus Nephritis (LN) patients is common and multifactorial. The aim of this study was to evaluate the bone status of newly diagnosed LN patients and their correlation with inflammatory factors involved in LN physiopathology.

Methods

We studied 15 pre-menopausal patients with ≤2 months of diagnosed SLE and LN. Patients with prior kidney or bone disease were excluded. In addition to biochemical evaluation (including 25-hydroxyvitamin D₃ [25(OH)D] and Monocyte Chemotactic Protein (MCP1) dosage), we performed bone biopsies followed by osteoblast culture, histomorphometric and immunohistochemistry analysis.

Results

LN patients presented a mean age of 29.5±10 years, a proteinuria of 4.7±2.9 g/day and an estimated glomerular filtration rate (GFR) of 37(31–87) ml/min/1,73 m². They were on glucocorticoid therapy for 34±12 days. All patients presented vitamin D insufficiency (9.9±4.4 ng/ml, range 4–20). Urinary MCP1 correlated negatively with 25(OH)D (r = −0.53, p = 0.003) and positively with serum deoxypyridinoline (r = 0.53, p = 0.004). Osteoblasts isolated from LN bone biopsies presented a significantly higher expression of MCP-1 when compared to controls (32.0.±9.1 vs. 22.9±5.3 mean fluorescence intensities, p = 0.01). LN patients presented a significantly reduced osteoid volume, osteoid thickness, osteoid surface, mineralization surface and bone formation rate, associated with an increased eroded surface and osteoclast surface. Patient’s bone specimens demonstrated a reduced immunostaining for osteoprotegerin (0.61±0.82 vs. 1.08±0.50%, p = 0.003), and an increased expression of Receptor Activator of NF-κB ligand (RANKL) (1.76±0.92 vs. 0.41±0.28%, p<0.001) when compared to controls.

Discussion

Newly diagnosed LN patients presented a significant disturbance in bone metabolism, characterized by an impaired bone formation and mineralization, associated with an increase in resorption parameters. Glucocorticoid use, vitamin D insufficiency and inflammation might be involved in the physiopathology of bone metabolism disturbance.     

Multilineage potential of adult human mesenchymal stromal cells derived from bone marrow of patients with polycytaemia vera

Human mesenchymal stromal cells were isolated from the bone marrow of patients with polycyteamia vera (the myeloproliferative disorder) with the aim to characterize the properties of the mesenchymal stromal cells originating from the pathologically affected bone marrow. Their in vitro growth and potential to differentiate were determined. Isolated mesenchymal stromal cells were able to differentiate into three mesenchymal lineages under appropriate cultivation conditions.