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Background and Purpose: Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca(2+) store in uterine activity.Experimental Approach: We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca(2+) concentration ([Ca(2+) ](c) ) in longitudinal smooth muscle of the mouse uterus.Key Results: The mitochondrial agents rotenone, carbonylcyanide-3-chlorophenylhydrazone (CCCP), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157) and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared with those of SER inhibitors cyclopiazonic acid (CPA), 2-amino ethoxyphenylborate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca(2+) ](c) or depolarization/transient hyperpolarization and transient increase in [Ca(2+) ](c) for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca(2+) ](c) . CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca(2+) ](c) . Application of a range of K(+) channel blockers indicated a role of Ca(2+) -activated K(+) (K(Ca) ) channels in the CCCP- and CGP37157-induced actions.Conclusions and Implications: Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca(2+) ](c) and/or Ca(2+) influx. [ABSTRACT FROM AUTHOR]

The nitrogen-related PTS Ntr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EI Ntr , NPr and EIIA Ntr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EI Ntr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIA Ntr . The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EI Ntr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTS Ntr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIA Ntr , regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIA Ntr and its target proteins is usually regulated by the EIIA Ntr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIA Ntr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIA Ntr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIA Ntr binding partners., Competing Interests: Declaration of competing interest Authors have no conflict of interest to declare., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors., (Copyright © 2020 Gerhardt et al.)

The intense use of herbicides for weed control in agriculture causes selection pressure on soil microbiota and water ecosystems, possibly resulting in changes to microbial processes, such as biogeochemical cycles. These xenobiotics may increase the production of reactive oxygen species and consequently affect the survival of microorganisms, which need to develop strategies to adapt to these conditions and maintain their ecological functionality. This study analyzed the adaptive responses of bacterial isolates belonging to the same species, originating from two different environments (water and soil), and subjected to selection pressure by herbicides. The effects of herbicide Callisto and its active ingredient, mesotrione, induced different adaptation strategies on the cellular, enzymatic, and structural systems of two Bacillus megaterium isolates obtained from these environments. The lipid saturation patterns observed may have affected membrane permeability in response to this herbicide. Moreover, this may have led to different levels of responses involving superoxide dismutase and catalase activities, and enzyme polymorphisms. Due to these response systems, the strain isolated from water exhibited higher growth rates than did the soil strain, in evaluations made in oligotrophic culture media, which would be more like that found in semi-pristine aquatic environments. The influence of the intracellular oxidizing environments, which changed the mode of degradation of mesotrione in our experimental model and produced different metabolites, can also be observed in soil and water at sites related to agriculture. Since the different metabolites may present different levels of toxicity, we suggest that this fact should be considered in studies on the fate of agrochemicals in different environments.

The carbohydrate-uptake phosphorelay PTS system plays a key role in metabolic regulation in Bacteria controlling the utilization of secondary carbon sources. Some bacteria, such as Escherichia coli, encode a paralogous system named PTS Ntr (nitrogen related PTS). PTS Ntr is composed of EI Ntr (ptsP), NPr (ptsO), and EIIA Ntr (ptsN). These proteins act as a phosphorelay system from phosphoenolpyruvate to EI Ntr , NPr and them to EIIA Ntr . PTS Ntr is not involved in carbohydrate uptake and it may be dedicated to performing regulatory functions. The phosphorylation state of EI Ntr is regulated by allosteric binding of glutamine and 2-oxoglutarate, metabolites whose intracellular levels reflect the nitrogen status. Although PTS Ntr is designated as having nitrogen-sensory properties, no major effect of this system on nitrogen regulation has been described in E. coli. Here we show that an E. coli ptsN deletion mutant has impaired growth in minimal medium. Proteome analysis of the ∆ptsN strain under different nitrogen regimes revealed no involvement in regulation of the canonical nitrogen regulatory (Ntr) system. The proteomic data support the conclusion that ptsN is required to balance the activities of the sigma factors RpoS and RpoD in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed., Significance: The nitrogen related PTS Ntr phosphorelay system has been hypothesized to participate in the control of nitrogen metabolism. Here we used a proteomics approach to show that an Escherichia coli ptsN null strain, which misses the final module of PTS Ntr phosphorelay, has no significant effects on nitrogen metabolism under different nitrogen regimes. We noted that ptsN is required for fitness under minimal medium and for the proper balance between RpoS and sigma 70 activities in such way that, in the absence of ptsN, RpoS-dependent genes are preferentially expressed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Herbicides are continuously used to minimize the loss of crop productivity in agricultural environments. They can, however, cause damage by inhibiting the growth of microbiota via oxidative stress, due to the increased production of reactive oxygen species (ROS). Cellular responses to ROS involve the action of enzymes, including superoxide dismutase (SOD) and catalase (CAT). The objective of this study was to evaluate adaptive responses in Escherichia coli K-12 to paraquat, the active ingredient in the herbicide Gramoxone®. Mutant bacterial strains carrying deletions in genes encoding Mn-SOD (sodA) and Fe-SOD (sodB) were used and resulted in distinct levels of hydrogen peroxide production, interference in malondialdehyde, and viability. Mutations also resulted in different levels of interference with the activity of CAT isoenzymes and in the inactivation of Cu/Zn-SOD activity. These mutations may be responsible for metabolic differences among the evaluated strains, resulting in different patterns of antioxidative responses, depending on mutation background. While damage to the ΔsodB strain was minor at late log phase, the reverse was true at mid log phase for the ΔsodA strain. These results demonstrate the important role of these genes in defense against oxidative stress in different periods of growth. Furthermore, the lack of Cu/Zn-SOD activity in both mutant strains indicated that common metal cofactors likely interfere in SOD activity regulation. These results also indicate that E. coli K-12, a classical non-environmental strain, constitutes a model of phenotypic plasticity for adaptation to a redox-cycling herbicide through redundancy of different isoforms of SOD and CAT enzymes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nitrogen is needed for the biosynthesis of biomolecules including proteins and nucleic acids. In the absence of fixed nitrogen prokaryotes such as E. coli immediately ceases growth. Ammonium is the preferred nitrogen source for E. coli supporting the fastest growth rates. Under conditions of ammonium limitation, E. coli can use alternative nitrogen sources to supply ammonium ions and this reprogramming is led by the induction of the NtrC regulon. Here we used label free proteomics to determine the dynamics of E. coli proteins expression in response to ammonium starvation in both the short (30min) and the longer (60min) starvation. Protein abundances and post-translational modifications confirmed that activation of the NtrC regulon acts as the first line of defense against nitrogen starvation. The ribosome inactivating protein Rmf was induced shortly after ammonium exhaustion and this was preceded by induction of other ribosome inactivating proteins such as Hpf and RaiA supporting the hypothesis that ribosome shut-down is a key process during nitrogen limitation stress. The proteomic data revealed that growth arrest due to nitrogen starvation correlates with the accumulation of proteins involved in DNA condensation, RNA and protein catabolism and ribosome hibernation. Collectively, these proteome adaptations will result in metabolic inactive cells which are likely to exhibit multidrug tolerance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na(+) or oscillatory currents were detected. Ca(2+)-free and Ba(2+)-containing solutions failed to demonstrate a Ca(2+) current during the interspike interval, although a Ca(2+) current was activated at membrane potentials positive to -40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K(+) conductance during the interspike interval. Ca(2+)-activated K(+), inward rectifier and low-TEA (10 muM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca(2+) current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na(+) current and a high TEA-sensitive K(+) current. In contrast with rats, a persistent Ca(2+) current is not involved., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Tissue transglutaminase (tTG) has recently been identified as the autoantigen recognized by endomysial antibodies in celiac disease (CD) patients and this has permitted the use of an ELISA test to detect the presence in the serum of autoantibodies specific for the diagnosis of CD., Aim: We report two cases of anti-tTG positivity in patients with non-Hodgkin's lymphoma (NHL) without evidence of CD., Case Reports: Both patients were males aged 67 and 69 years respectively; both were hospitalized for fever and peripheral adenopathy. Lymph node histology showed an immunoblastic high-grade T-cell NHL at the IVth the stage of disease in both cases. They were included in a multicenter study on the association between CD and NHL. Serological screening for CD showed the presence of serum anti-tTG antibodies, with values within the range of those recorded in untreated CD patients in our laboratory; however, both patients had negative anti-endomysial antibodies and in both cases intestinal histology showed normal mucosa with villi and crypts of normal height and depth (villi/crypts ratio > or = 2.5, within the range of normal subjects for our laboratory), and no increase in intraepithelial lymphocytes. The HLA phenotype was obtained giving the following antigens: Case 1: A 3, A 24(9), B 22, B 35, BW 6, DR 1, DR 11(5), DQ 3, DR 52. Case 2: A 2, A 3, B 51(5), B 8, BW 4, BW 6, DR B1*02, DR B1*03, DR B3*01. Both subjects were also positive for serum anti-smooth muscle antibodies and one for antinuclear antibodies., Conclusions: (1) Serum anti-tTG positivity can be found in subjects with NHL without CD and the real frequency of these 'false positives' must be investigated both in subjects with lymphoproliferative disorders and in patients with autoimmune diseases. (2) In patients with NHL, without CD, anti-tTG positivity may be unassociated with EmA positivity and the biological significance of this finding must be clarified., (Copyright 2000 S. Karger AG, Basel)

A case of an actinomycotic brain abscess in an adult patient apparently free of systemic infection is presented. The rarity of this etiology of brain abscess is underlined. The patient was treated by complete surgical excision and prolonged antibiotic therapy with penicillin. The authors stress the importance of taking into account this infrequent cause of brain abscess, the necessity of radical surgical treatment followed by intensive, prolonged and adequate antibiotic therapy, as the only means of reducing the high mortality rate.