Gonzalo Costa

Transcriptional and proteomic stress responses of a soil bacterium Bacillus cereus to nanosized zero-valent iron (nZVI) particles

Nanosized zero valent iron (nZVI) is emerging as an option for treating contaminated soil and groundwater even though the potentially toxic impact exerted by nZVI on soil microorganisms remains uncertain. In this work, we focus on nanotoxicological studies performed in vitro using commercial nZVI and one common soil bacterium (Bacillus cereus). Results showed a negative impact of nZVI on B. cereus growth capability, consistent with the entrance of cells in an early sporulation stage, observed by TEM. Despite no changes at the transcriptional level are detected in genes of particular relevance in cellular activity (narG, nirS, pykA, gyrA and katB), the proteomic approach used highlights differentially expressed proteins in B. cereus under nZVI exposure. We demonstrate that proteins involved in oxidative stress-response and tricarboxilic acid cycle (TCA) modulation are overexpressed; moreover proteins involved in motility and wall biosynthesis are repressed. Our results enable to detect a molecular-level response as early warning signal, providing new insight into first line defense response of a soil bacterium after nZVI exposure.

Endothelium-dependent relaxation to acetylcholine in bovine oviductal arteries: mediation by nitric oxide and changes in apamin-sensitive K+ conductance

1 Mechamisms underlying the relaxant response to acetylcholine (ACh) were examined in bovine oviductal arteries (o.d. 300–500 μm and i.d. 150–300μm) in vitro. Vascular rings were treated with indomethacin (10 μm) to prevent the effects of prostaglandins. 2 ACh elicited a concentration‐related relaxation in ring segments precontracted with noradrenaline (NA), which was abolished by endothelium denudation. 3 The ACh‐induced relaxation was attenuated but not abolished by NG‐nitro‐L‐arginine (l‐NOARG, 1 μm‐1 mM), an inhibitor of nitric oxide (NO) formation. The inhibition caused by L‐NOARG (10 μm) was reversed by addition of excess of L‐arginine but not D‐arginine (1 mM). 4 In high K+ (40–60 mM)‐contracted rings, ACh was a much less effective vasodilator and its relaxant response was completely abolished by L‐NOARG (100 μm). 5 In NA (10 μm)‐contracted rings, ACh induced sustained and concentration‐dependent increases in cyclic GMP, which were reduced below basal values by L‐NOARG (100 μm), while potent relaxation persisted. Similar increases in cyclic GMP were evoked by ACh in high K+ (50 mM)‐treated arteries and under these conditions, both cyclic GMP accumulation and relaxation were L‐NOARG‐sensitive. 6 S‐nitroso‐L‐cysteine (NC), a proposed endogenous precursor of endothelial NO, also induced cyclic GMP accumulation in NA‐contracted oviductal arteries. 7 Methylene blue (MB, 10 μm), a proposed inhibitor of soluble guanylate cyclase, inhibited both endothelium‐dependent relaxation to ACh and endothelium‐independent response to exogenous NO, whereas relaxation to NC remained unaffected. 8 The L‐NOARG‐resistant response to ACh was not affected by either ouabain (0.5 mM), glibenclamide (3 μm), tetraethylammonium (TEA, 1 mM) or charybdotoxin (50 nM), but was selectively blocked by apamin (0.1‐1 μm). However, apamin did not inhibit either relaxation to ACh in high K+‐contracted rings or endothelium‐independent relaxation to either NO or NC. 9 Apamin and MB inhibited ACh‐induced relaxation in an additive fashion, suggesting the involvement of two separate modulating mechanisms. 10 These results suggest that ACh relaxes bovine oviductal arteries by the release of two distinct endothelial factors: a NO‐like substance derived from L‐arginine, which induces cyclic GMP accumulation in smooth muscle, and another non‐prostanoid factor acting by hyperpolarization mechanisms through alterations in apamin‐sensitive K+ conductance.

Molecular stress responses to nano-sized zero-valent iron (nZVI) particles in the soil bacterium Pseudomonas stutzeri.

Nanotoxicological studies were performed in vitro using the common soil bacterium Pseudomonas stutzeri to assess the potentially toxic impact of commercial nano-sized zero-valent iron (nZVI) particles, which are currently used for environmental remediation projects. The phenotypic response of P. stutzeri to nZVI toxicity includes an initial insult to the cell wall, as evidenced by TEM micrographs. Transcriptional analyses using genes of particular relevance in cellular activity revealed that no significant changes occurred among the relative expression ratios of narG, nirS, pykA or gyrA following nZVI exposure; however, a significant increase in katB expression was indicative of nZVI-induced oxidative stress in P. stutzeri. A proteomic approach identified two major defence mechanisms that occurred in response to nZVI exposure: a downregulation of membrane proteins and an upregulation of proteins involved in reducing intracellular oxidative stress. These biomarkers served as early indicators of nZVI response in this soil bacterium, and may provide relevant information for environmental hazard assessment.

Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

1 To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO‐mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2 NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg−1 protein min−1): urethra (33 ± 3.3), detrusor (13.1 ± 1.1) and ureter (1.5 ± 0.2). No activity was detected in the particulate fraction of any region. 3 NOS activity was dependent on Ca2+‐calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4 NOS activity was inhibited to a greater extent by NG‐nitro‐l‐arginine (l‐NOARG) and its methyl ester (l‐NAME) than by NG‐monomethyl‐l‐arginine (l‐NMMA), while 7‐nitroindazole (7‐NI) was a weak inhibitor and L‐cannavine had no effect. 5 Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin‐sensitive manner, suggesting feedback inhibition of NOS by NO. 6 EFS induced prominent NO‐mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L‐NOARG = L‐NAME > 7‐NI > L‐NMMA. 7 In conclusion, we have demonstrated the presence of NO‐synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO‐mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.

Integrating classical and molecular approaches to evaluate the impact of nanosized zero-valent iron (nZVI) on soil organisms.

Nanosized zero-valent iron (nZVI) is a new option for the remediation of contaminated soil and groundwater, but the effect of nZVI on soil biota is mostly unknown. In this work, nanotoxicological studies were performed in vitro and in two different standard soils to assess the effect of nZVI on autochthonous soil organisms by integrating classical and molecular analysis. Standardised ecotoxicity testing methods using Caenorhabditis elegans were applied in vitro and in soil experiments and changes in microbial biodiversity and biomarker gene expression were used to assess the responses of the microbial community to nZVI. The classical tests conducted in soil ruled out a toxic impact of nZVI on the soil nematode C. elegans in the test soils. The molecular analysis applied to soil microorganisms, however, revealed significant changes in the expression of the proposed biomarkers of exposure. These changes were related not only to the nZVI treatment but also to the soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis. Furthermore, due to the temporal shift between transcriptional responses and the development of the corresponding phenotype, the molecular approach could anticipate adverse effects on environmental biota.

Residual impact of aged nZVI on heavy metal-polluted soils.

In the present study, the residual toxicity and impact of aged nZVI after a leaching experiment on heavy metal (Pb, Zn) polluted soils was evaluated. No negative effects on physico-chemical soil properties were observed after aged nZVI exposure. The application of nZVI to soil produced a significant increase in Fe availability. The impact on soil biodiversity was assessed by fluorescence in situ hybridization (FISH). A significant effect of nZVI application on microbial structure has been recorded in the Pb-polluted soil nZVI-treated. Soil bacteria molecular response, evaluated by RT-qPCR using exposure biomarkers (pykA, katB) showed a decrease in the cellular activity (pykA) due to enhanced intracellular oxidative stress (katB). Moreover, ecotoxicological standardised test on Caenorhabditis elegans (C. elegans) showed a decrease in the growth endpoint in the Pb-polluted soil, and particularly in the nZVI-treated. A different pattern has been observed in Zn-polluted soils: no changes in soil biodiversity, an increase in biological activity and a significant decrease of Zn toxicity on C. elegans growth were observed after aged nZVI exposure. The results reported indicated that the pollutant and its nZVI interaction should be considered to design soil nanoremediation strategies to immobilise heavy metals.

Interstitial cells of Cajal in the urethra are cGMP-mediated targets of nitrergic neurotransmission

While interstitial cells of Cajal (ICC) in the urethra respond to nitric oxide (NO) donors by increasing cGMP, it remains unclear whether urethral ICC are functionally innervated by nitrergic nerves. We have addressed this issue in the rat and sheep urethra, where cGMP production and relaxation were compared in preparations subjected to electrical field stimulation (EFS; 2 Hz, 4 min) of nitrergic nerves or to exogenous S-nitroso-L-cysteine (SNC; 0.1 mM, 4 min). Upon EFS, cGMP immunoreactivity (cGMP-ir) was observed in both smooth muscle cells (SMC) and in spindle-shaped cells that contained c-kit and vimentin, features of ICC. Similarly, cGMP-ir was preferentially, but inconsistently, found in ICC of the outer muscle layer on exposure to SNC. We found separate functional groups of ICC within the urethra. Thus only ICC present in the muscle layers (ICC-M) but not those in the serosa (ICC-SR) and lamina propia (ICC-LP) seem to be specifically influenced by activation of neuronal NO synthase (nNOS). Thus the increase in cGMP-ir in the ICC-M induced by EFS was prevented by Nomega-nitro-L-arginine and ODQ. Urethral ICC did not express nNOS, although they were closely associated with nitrergic nerves. cGMP-ir was also present in the urothelium (in the rat but not in the sheep) and the vascular endothelium but not in neural structures, such as the nerve trunks and nerve terminals. Together, these results suggest a model of parallel innervation in which both SMC and ICC-M are effectors of nerve-released NO in the urethra.

Local regulation of oviductal blood flow.

1. Blood flow to the oviduct is implicated in the genesis and maintenance of oviductal fluid, in this way contributing to the creation of an adequate medium for ovum/embryo physiology. Therefore, factors controlling the tone of the vessels supplying the oviduct would be expected to affect its luminal environment. In addition, cyclic changes in oviductal blood flow have been suggested to have mechanical functions in the transport of the ovum/embryo. 2. The vascular supply to the oviduct has a prominent adrenergic vasomotor control. A dense adrenergic innervation, together with the presence of a predominant population of alpha(1)-adrenoceptors, provides a contractile regulatory mechanism of oviductal blood flow. No evidence is available on the presence of beta-adrenoceptors. The scanty cholinergic innervation of mammalian oviduct is mainly confined to the vessels, where acetylcholine (ACh) has a vasodilatory effect by releasing endothelium-derived relaxing factors. 3. The presence of nerves containing neuropeptides has been shown in the oviduct. Specifically, a high density of neuropeptide Y- and vasointestinal peptide-containing nerve fibers has been found in relation to blood vessels, but their role in the neutral control of the oviduct blood flow remains to be established. To date, it is not known whether or not oviductal blood vessels receive perivascular nitrergic nerves. 4. Relaxing and contracting factors derived from endothelium also seem to have a modulatory role on oviductal vascular tone. Neurotransmitters or autacoids, such as ACh and histamine, acting on endothelial receptors, release nitric oxide (NO), which relaxes oviductal arteries through guanylyl cyclase activation and accumulation of cyclic GMP. In addition, the release of an endothelium-derived hyperpolarizing factor (EDHF), distinct from NO, by ACh has been shown in oviductal arteries. It acts through the opening of low-conductance Ca(2+)-activated K+ channels leading to hyperpolarization and relaxation. Furthermore, potent and long-lasting contractions induced by the endothelium-derived contractile factor, endothelin (ET), points to its role in the long-term regulation of oviductal vascular tone. 5. A particularly high density of 5-hydroxytryptamine (5-HT) and histamine, present in mast cells clustered in the vicinity of blood vessels, has been described in the oviduct. It is known that histamine elicits a relaxation of oviductal arteries that is partially endothelium-dependent and mediated by the activation of H1-receptors. The implication of histamine in both the increase in blood flow and edema around ovulation, as well as the existence of a functional antagonism between histamine and 5-HT in the regulation of oviductal blood flow, await further investigation. 6. Other factors, such as relaxing and contracting cyclooxygenase-derived products, may also participate in the modulation of blood flow to the oviduct. 7. An overall endocrine regulation of the oviductal vascular supply exists, acting by both direct effects on smooth muscle and modulation of neural and autocrine factors. This control enables cyclic changes in blood flow to the oviduct that are tightly coupled to the reproductive functions of the tube.

Impact of Ag and Al2O3 nanoparticles on soil organisms: In vitro and soil experiments

In vitro analyses were conducted to assess the impact of Al2O3 and Ag nanoparticles on two common soil bacteria, Bacillus cereus and Pseudomonas stutzeri. Al2O3 nanoparticles did not show significant toxicity at any dose or time assayed, whereas exposure to 5 mg L(-1) Ag nanoparticles for 48 h caused bactericidal effects. Moreover, alterations at the morphological level were observed by transmission electron microscopy (TEM); Ag but not Al2O3 nanoparticles evoked the entrance of B. cereus cells in an early sporulation stage and both nanoparticles penetrated P. stutzeri cells. At the molecular level, a dramatic increase (8.2-fold) in katB gene expression was found in P. stutzeri following Al2O3 nanoparticles exposure, indicative of an oxidative stress-defence system enhancement in this bacterium. In the microcosm experiment, using two different natural soils, Al2O3 or Ag nanoparticles did not affect the Caenorhabditis elegans toxicity endpoints growth, survival, or reproduction. However, differences in microbial phylogenetic compositions were detected by fluorescence in situ hybridization (FISH). The use of katB- and pykA-based sequences showed that the microbial transcriptional response to nanoparticle exposure decreased, suggesting a decrease in cellular activity. These changes were attributable to both the nanoparticles treatment and soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis.

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoine‐1‐oxyl 3‐oxide (carboxy‐PTIO), the specific guanylate cyclase inhibitor 1H‐[1,2,4]‐oxadiazole‐[4,3‐a]‐quinoxalin‐1‐one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 μM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml−1) inhibited NO‐mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy‐PTIO (0.1–0.5 mM) inhibited NO‐mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml−1). Relaxations to NO were reduced, but not abolished, by ODQ (10 μM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5′‐dithio‐bis (2‐nitrobenzoic acid) (DTNB, 0.5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N‐ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L‐cysteine (L‐cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD‐independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open.