The Glutathione Metabolite γ-Glutamyl-Glutamate Partially Activates Glutamate NMDA Receptors in Central Neurons With Higher Efficacy for GluN2B-Containing Receptors

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced.

Release of HIV-1 particles from the viral compartment in macrophages requires an associated cytoskeleton and is driven by mechanical constraints

A defining feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named VCC (virus-containing compartments) that is connected to the extracellular medium. The newly formed viral progeny pinches of the membrane and accumulates in the lumen of the VCC. While HIV budding has been extensively studied, very little is known about how viral particles present in the lumen of VCC are released in the extracellular medium. Here we show that the actin dynamics are critical for this process by combining ultrastructural analyses, time-lapse microscopy and perturbations of the actin cytoskeleton. We found that jasplakinolide, which stabilizes actin fibres, inhibited viral release from HIV-1-infected macrophages, but not from infected HeLa cells. Furthermore, in jasplakinolide-treated macrophages, VCC became scattered and no longer co-localized with the integrin CD18, nor the phosphorylated form of the focal adhesion kinase PYK2. Inhibition of PYK2 activity in infected macrophages promoted intracellular retention of viral particles in VCC that were no longer connected to the plasma membrane. Finally, we stimulated the rapid release of viral particles from the VCC by subjecting infected macrophages to frustrated phagocytosis. As macrophages spread on IgG-coated glass surfaces, VCC rapidly migrated to the basal membrane and released their viral content in the extracellular medium, which required their association with CD18 and the actin cytoskeleton. These results highlight that VCC trafficking and virus release are intimately linked to the reorganization of the macrophage actin cytoskeleton in response to external physical cues, suggesting that it might be regulated in tissues by the mechanical stress to which these cells are exposed.

Biosynthesis Pathways, Transport Mechanisms and Biotechnological Applications of Fungal Siderophores

Iron (Fe) is the fourth most abundant element on earth and represents an essential nutrient for life. As a fundamental mineral element for cell growth and development, iron is available for uptake as ferric ions, which are usually oxidized into complex oxyhydroxide polymers, insoluble under aerobic conditions. In these conditions, the bioavailability of iron is dramatically reduced. As a result, microorganisms face problems of iron acquisition, especially under low concentrations of this element. However, some microbes have evolved mechanisms for obtaining ferric irons from the extracellular medium or environment by forming small molecules often regarded as siderophores. Siderophores are high affinity iron-binding molecules produced by a repertoire of proteins found in the cytoplasm of cyanobacteria, bacteria, fungi, and plants. Common groups of siderophores include hydroxamates, catecholates, carboxylates, and hydroximates. The hydroxamate siderophores are commonly synthesized by fungi. L-ornithine is a biosynthetic precursor of siderophores, which is synthesized from multimodular large enzyme complexes through non-ribosomal peptide synthetases (NRPSs), while siderophore-Fe chelators cell wall mannoproteins (FIT1, FIT2, and FIT3) help the retention of siderophores. S. cerevisiae, for example, can express these proteins in two genetically separate systems (reductive and nonreductive) in the plasma membrane. These proteins can convert Fe (III) into Fe (II) by a ferrous-specific metalloreductase enzyme complex and flavin reductases (FREs). However, regulation of the siderophore through Fur Box protein on the DNA promoter region and its activation or repression depend primarily on the Fe availability in the external medium. Siderophores are essential due to their wide range of applications in biotechnology, medicine, bioremediation of heavy metal polluted environments, biocontrol of plant pathogens, and plant growth enhancement.

Systematic Review of Calcium Channels and Intracellular Calcium Signaling: Relevance to Pesticide Neurotoxicity

Pesticides of different chemical classes exert their toxic effects on the nervous system by acting on the different regulatory mechanisms of calcium (Ca2+) homeostasis. Pesticides have been shown to alter Ca2+ homeostasis, mainly by increasing its intracellular concentration above physiological levels. The pesticide-induced Ca2+ overload occurs through two main mechanisms: the entry of Ca2+ from the extracellular medium through the different types of Ca2+ channels present in the plasma membrane or its release into the cytoplasm from intracellular stocks, mainly from the endoplasmic reticulum. It has also been observed that intracellular increases in the Ca2+ concentrations are maintained over time, because pesticides inhibit the enzymes involved in reducing its levels. Thus, the alteration of Ca2+ levels can lead to the activation of various signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. In this review, we also discuss some proposed strategies to counteract the detrimental effects of pesticides on Ca2+ homeostasis.

Effect of Exogenous and Endogenous Sulfide on The Production and The Export of Methylmercury by Sulfate Reducing Bacteria

Mercury (Hg) is a global pollutant of environmental and health concern; its methylated form, methylmercury (MeHg) is a potent neurotoxin. Sulfur-containing molecules play a role in MeHg production by microorganisms. While sulfides are considered to limit Hg methylation, sulfate and cysteine were shown to favor this process. However, these two forms can be endogenously converted by microorganisms into sulfide. Here, we explore the effect of sulfide (produced by the cell or supplied exogenously) on Hg methylation. For this purpose, Pseudodesulfovibrio hydrargyri BerOc1 was cultivated in non-sulfidogenic conditions with addition of cysteine and sulfide as well as in sulfidogenic conditions. We report that Hg methylation depends on sulfide concentration in the culture rather than on the initial form of sulfur (cysteine, sulfide or sulfate) added, and was independent of hgcA expression. Interestingly, MeHg production was maximal at 0.1-0.5 mM of sulfides. Besides, a strong positive correlation between MeHg in the extracellular medium and the increase of sulfide concentrations was observed, suggesting a facilitated MeHg export with sulfide and/or higher desorption from the cell. We demonstrate that sulfides (exogenous or endogenous) play a key role in controlling mercury methylation, and should be considered when investigating the impact of Hg on natural environments.

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca2+ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca2+ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca2+ signals by astrocytes occurs through the release of Ca2+ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca2+ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca2+ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.

A hemolysin secretion pathway-based novel secretory expression platform for efficient manufacturing of tag peptides and anti-microbial peptides in Escherichia coli

Background Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. Results In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes. Conclusions The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents. Graphical Abstract

A new approach to the determination of tubular membrane capacitance: passive membrane electrical properties under reduced electrical conductivity of the extracellular solution

The tubular system of cardiomyocytes plays a key role in excitation-contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances by electrophysiological measurements are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separately capacitances of the tubular and surface membranes from altered capacitive current responses to subthreshold voltage-clamped rectangular pulses. Based on the analysis of the time course of capacitive current, computational relations were derived which allow to quantify elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. In addition, it might be possible to identify changes in both surface and tubular membrane capacitances caused by various interventions. Preliminary experiments in rat ventricular cardiomyocytes (n = 10) resulted in values of the surface and tubular capacitances 72.3 ± 16.4 and 42.1 ± 14.7 pF, respectively, implying the fraction of tubular capacitance/area of 0.36 ± 0.08. We conclude that the newly proposed method provides results comparable to those reported in literature and, in contrast to the currently used methods, enables repetitive evaluation of parameters describing the surface and tubular membranes. It may be used to study alterations of the tubular system resulting from various interventions including associated cardiac pathologies.

Probenecid affects sarcoplasmic reticulum Ca2+ release and depresses contractile activation in mouse skeletal muscle

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

Exosome-Mediated Insulin Delivery for the Potential Treatment of Diabetes Mellitus

Exosomes are extracellular nanovesicles between 30 and 150 nm that serve as essential messengers for different biological signaling and pathological processes. After their discovery, a wide range of applications have been developed, especially in therapeutic drug delivery. In this context, the aim of this work was to test the efficiency of exosome-mediated human insulin delivery using exosomes extracted from three different cell lines: hepatocellular carcinoma (HepG2); primary dermal fibroblasts (HDFa) and pancreatic β cells (RIN-m); all are related to the production and/or the ability to sense insulin and to consequently regulate glucose levels in the extracellular medium. The obtained results revealed that the optimal insulin loading efficiency was achieved by a 200 V electroporation, in comparison with incubation at room temperature. Moreover, the maximum in vitro exosome uptake was reached after incubation for 6 h, which slightly decreased 24 h after adding the exosomes. Glucose quantification assays revealed that exosome-mediated incorporation of insulin presented significant differences in HDFa and HepG2 cells, enhancing the transport in HDFa, in comparison with free human insulin effects in the regulation of extracellular glucose levels. No significant differences were found between the treatments in RIN-m cells. Hence, the results suggest that exosomes could potentially become a valuable tool for stable and biocompatible insulin delivery in diabetes mellitus treatment alternatives.