Cecilia I. Muglia

Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.

Glutathione Plays a Fundamental Role in Growth and Symbiotic Capacity of Sinorhizobium meliloti

Rhizobia form a symbiotic relationship with plants of the legume family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We have examined the importance of glutathione (GSH) during free-living growth and symbiosis of Sinorhizobium meliloti. An S. meliloti mutant strain (SmgshA) which is unable to synthesize GSH due to a gene disruption in gshA, encoding the enzyme for the first step in the biosynthesis of GSH, was unable to grow under nonstress conditions, precluding any nodulation. In contrast, an S. meliloti strain (SmgshB) with gshB, encoding the enzyme involved in the second step in GSH synthesis, deleted was able to grow, indicating that gamma-glutamylcysteine, the dipeptide intermediate, can partially substitute for GSH. However, the SmgshB strain showed a delayed-nodulation phenotype coupled to a 75% reduction in the nitrogen fixation capacity. This phenotype was linked to abnormal nodule development. Both the SmgshA and SmgshB mutant strains exhibited higher catalase activity than the wild-type S. meliloti strain, suggesting that both mutant strains are under oxidative stress. Taken together, these results show that GSH plays a critical role in the growth of S. meliloti and during its interaction with the plant partner.

Vanadium and cancer treatment: antitumoral mechanisms of three oxidovanadium(IV) complexes on a human osteosarcoma cell line.

We report herein the antitumor actions of three oxidovanadium(IV) complexes on MG-63 human osteosarcoma cell line. The three complexes: VO(oda), VO(oda)bipy and VO(oda)phen (oda=oxodiacetate), caused a concentration dependent inhibition of cell viability. The antiproliferative action of VO(oda)phen could be observed in the whole range of concentrations (at 2.5 μM), while VO(oda)bipy and VO(oda) showed a decrease of cell viability only at higher concentrations (at 50 and 75 μM, respectively) (p<0.01). Moreover, VO(oda)phen caused a decrease of lysosomal and mitochondrial activities at 2.5 μM, while VO(oda) and VO(oda)bipy affected neutral red uptake and mitochondrial metabolism at 50 μM (p<0.01). On the other hand, no DNA damage studied by the Comet assay could be observed in MG-63 cells treated with VO(oda) at 2.5-10 μM. Nevertheless, VO(oda)phen and VO(oda)bipy induced DNA damage at 2.5 and 10 μM, respectively (p<0.01). The generation of reactive oxygen species increased at 10 μM of VO(oda)phen and only at 100 μM of VO(oda) and VO(oda)bipy (p<0.01). Besides, VO(oda)phen and VO(oda)bipy triggered apoptosis as determined by externalization of the phosphatidylserine. The determination of DNA cleavage by agarose gel electrophoresis showed that the ability of VO(oda)(bipy) is similar to that of VO(oda), while VO(oda)(phen) showed the highest nuclease activity in this series. Overall, our results showed a good relationship between the bioactivity of the complexes and their structures since VO(oda)phen presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)bipy and then by VO(oda) according to the number of intercalating heterocyclic moieties.

Antitumor properties of a vanadyl(IV) complex with the flavonoid chrysin [VO(chrysin)2EtOH]2 in a human osteosarcoma model: the role of oxidative stress and apoptosis.

Flavonoids, a polyphenolic compound family, and the vanadium compounds have interesting biological, pharmacological, and medicinal properties. We report herein the antitumor actions of the complex [VO(chrysin)2EtOH]2 (VOchrys) on the MG-63 human osteosarcoma cell line. Oxovanadium(IV), chrysin and VOchrys caused a concentration-dependent inhibition of cell viability. The complex was the strongest antiproliferative agent (p < 0.05). Cytotoxicity and genotoxicity studies also showed a concentration effect. Reactive oxygen species (ROS) and the alterations in the GSH/GSSG ratio underlie the main mechanisms of action of VOchrys. Additions of ROS scavengers (vitamin C plus vitamin E) or GSH to the viability experiments demonstrated beneficial effects (p < 0.01). Besides, the complex triggered apoptosis, disruption of the mitochondria membrane potential (MMP), increased levels of caspase 3 and DNA fragmentation measured by the sub-G1 peak in cell cycle arrest experiments (p < 0.01). Collectively, VOchrys is a cell death modulator and a promissory complex to be used in cancer treatments.

Glutathione produced by Rhizobium tropici is important to prevent early senescence in common bean nodules

In this paper, we examine the importance of glutathione in symbiosis, using a glutathione biosynthetic gshB mutant derived from Rhizobium tropici CIAT899, a common bean (Phaseolus vulgaris) endosymbiont. Plants infected with the mutant strain presented a delayed nodulation phenotype and a reduction in the dry weight of aerial part of plants, suggesting diminished nitrogen-fixation activity. In addition, bacterial gshB expression was assayed in wild-type infected nodules, during the different steps of nodulation, and found to increase in mature and early senescent nodules. Conspicuously, nodules induced by gshB mutant bacteria presented an early senescent pattern, which was associated with increased levels of superoxide accumulation. These results provide a direct evidence of the role of bacterial glutathione in protecting nodules from reactive oxygen species, which may determine nodule senescence.

Cytotoxicity of a Vanadyl(IV) Complex with a Multidentate Oxygen Donor in Osteoblast Cell Lines in Culture

Strong chelating ligands as oxodiacetate (oda) are model systems to study the process of metal trapping by living organisms. Vanadium compounds display interesting biological and pharmacological actions. In vertebrates, vanadium is stored mainly in bones. In the present study we report the effects of the complex of oda with vanadyl(IV) cation, VO(oda), on two osteoblast cell lines, one normal (MC3T3E1) and the other tumoral (UMR106). VO(oda) exerted cytotoxic actions in osteoblasts as it was determined through a dose-dependent decrease in cell proliferation, and morphological and actin alterations. The putative mechanisms underlying VO(oda) deleterious effects were also investigated. The complex increased the level of ROS which correlated with a decreased in GSH/GSSG ratio. Besides, VO(oda) induced a dissipation of the mitochondria membrane potential (MMP) and promoted an increase in ERK cascade phosphorylation, which is involved in the regulation of cellular death and survival. All the effects were more pronounced in MC3T3-E1 than in UMR106 cells. ERK activation was inhibited by PD98059, Wortmanin and the ROS scavenger NAC (N-acetyl cysteine). These results suggest that VO(oda) stimulated ERKs phosphorilation by induction of free radicals involving kinases upstream of ERK pathway. The inhibitory effect of the complex on cell proliferation was partially reversed in both cell lines by NAC. Moreover, PD98059 and Wortmanin also partially reversed the inhibition of cell proliferation in the tumoral osteoblasts. The use of specific inhibitors and ROS scavengers suggested the involvement of oxidative stress, MMP alterations and ERK pathway in the apoptotic actions of this complex.

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1−/−) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1−/− mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.

Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1

Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin‐1 (Gal‐1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro‐inflammatory cytokines, and Gal‐1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal‐1 binding and modulation of IECs. We found an enhanced binding of Gal‐1 to: (a) murine IECs exposed to IL‐1β, TNF, and IL‐13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal‐1 favored a tolerogenic micro‐environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase‐9 and modulation of Bcl‐2 protein family members. Our results showed that, when added in the presence of diverse pro‐inflammatory cytokines such as tumor necrosis factor (TNF), IL‐13 and IL‐5, Gal‐1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL‐10, IL‐25, and transforming growth factor (TGF‐β1). In conclusion, we found an augmented binding of Gal‐1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal‐1 concentration. These findings highlight the importance of the inflammatory micro‐environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal‐1 and its role in epithelial cell homeostasis. J. Cell. Physiol. 231: 1575–1585, 2016.

A galectin‐specific signature in the gut delineates Crohn's disease and ulcerative colitis from other human inflammatory intestinal disorders

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohns disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for β-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosal-associated galectins (Gal-1, -3, -4, -9) is dysregulated in inflamed tissues of IBD patients compared with non-inflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.

Spectroscopic and Thermal Behavior of Na6[(VO)3(P2O7)3]·7H2O

The electronic (absorption and reflectance) as well as the infrared and Raman spectra of Na6[(VO)3(P2O7)3]·7H2O were recorded and discussed. Its thermal behavior was investigated by means of TG and DTA measurements. After a two-step dehydration, the compound melts at 464°C and, apparently, depolymerization takes place during this process. The characteristics of the pyrolysis residues, collected at different temperatures, were spectroscopically investigated.