Cholinergic Pesticides

Neurotoxicity exerted by organophosphates and carbamate

Characterization of Myelin Sheath FoF1-ATP Synthase and its Regulation by IF1

FoF1-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional FoF1- ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV FoF1-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/ nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F1 (IF1), a small protein that binds the F1 moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF1 in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration

Efficiency of two different transfection reagents for use with human NTERA2 cells

The teratocarcinoma cell line NTERA2 is recently used in a wide range of researches (from developmental biology to toxicology, for their ability to be induced to neural differentiation. In order to study the genetic potential of these cells, it is needed to use methods for gene silencing and/or mRNA interference, allowing cell viability and further differentiation. To check these features, we simultaneously tested the transfection efficiency of NTERA2, A549 and HeLa cells with Metafectene PRO (Biontex, Germany) and another optimal transfection reagent currently used in our Laboratory, using as a reporter gene the DsRed2 vector (Clontech, Mountain View, CA). Under our culture conditions for NTERA2 and HeLa cells, Metafectene PRO transfection method was found to possess high throughput performance, that allows low concentration rate and low exposure time to excitation light source, thus reducing both toxicity and phototoxicity

Inactivation of Crotalus atrox venom hemorrhagic activity by direct current exposure using hens' egg assay.

The hemotoxic venoms of Viperidae and Crotalidae are responsible for most of the evenomations in the United States, West Africa, India, South-East Asia, New Guinea, and Latin America. We previously reported that a short exposure of Crotalus atrox venom to direct electric current (dc) from a low-voltage generator, in solution, causes consistent and irreversible inactivation of venom phospholipase A 2 and metalloproteases. Here we report by in vivo assay on chicken embryos at stage 18 of development according to Hamburger and Hamilton that the hemorrhagic activity of C. atrox venom is lost after exposure to dc (from low voltage). Venom was exposed to dc ranging between 0 and 1 mA. dc values above 0.7 mA abolished hemorrhage. Such in vivo data, showing that dc neutralizes C. atrox venom hemorrhagic activity suggest that a deeper knowledge is needed to understand the relationship among dc and biological matte

Evidence for ectopic aerobic ATP production on C6 glioma cell plasma membrane.

Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism, besides mitochondria, is located also on the plasma membrane of C6 glioma cells. An ATP synthesis coupled to oxygen consumption was measured in plasma membrane isolated from C6 cells, sensitive to common inhibitors of respiratory chain complexes, suggesting the involvement of a putative surface ATP synthase complex. Immunofluorescence imaging showed that Cytochrome c oxydase colocalized with WGA, a typical plasma membrane protein, on the plasma membrane of glioma cells. Cytochrome c oxydase staining pattern appeared punctuate, suggesting the intriguing possibility that the redox chains may be expressed in discrete sites on C6 glioma cell membrane. Data suggest that the whole respiratory chain is localized on C6 glioma cell surface. Moreover, when resveratrol, an ATP synthase inhibitor, was added to culture medium, a cytostatic effect was observed, suggesting a correlation among the ectopic ATP synthesis and the tumor growth. So, a potential direction for the design of new targets for future therapies may arise

Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles.

The Central Nervous System (CNS) function was shown to be fueled exclusively by oxidative phosphorylation (OXPHOS). This is in line with the sensitivity of brain to hypoxia, but less with the scarcity of the mitochondria in CNS. Consistently with the ectopic expression of FoF1-ATP synthase and the electron transfer chain in myelin, we have reported data demonstrating that isolated myelin vesicles (IMV) conduct OXPHOS. It may suggest that myelin sheath could be a site for the whole aerobic degradation of glucose. In this paper, we assayed the functionality of glycolysis and of TCA cycle enzymes in IMV purified from bovine forebrain. We found the presence and activity of all of the glycolytic and TCA cycle enzymes, comparable to those in mitochondria-enriched fractions, in the same experimental conditions. IMV also contain consistent carbonic anhydrase activity. These data suggest that myelin may be a contributor in energy supply for the axon, performing an extra-mitochondrial aerobic OXPHOS. The vision of myelin as the site of aerobic metabolism may shed a new light on many demyelinating pathologies, that cause an a yet unresolved axonal degeneration and whose clinical onset coincides with myelin development completion

Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed

The strategic alliance between clinical and molecular science in the war against SARS-CoV-2, with the rapid-diagnostics test as an indispensable weapon for front line doctors

Our work concerns the actual problem of spread of SARS- CoV-2 outbreak which requires fast and correct as possible answer. In current scenario, the need of rapid answer put away the imperative of proper methodology. We focus on the serogical immunoassay for diagnosis of Covid-19 as an important weapon not only for diagnostic purpose, but also for epidemiologic one. The right equilibrium between high speed, low cost and accuracy is obtained with easy-to-use decentralized point-of-care test as the colloidal gold-based immunochromatographic strip assay which detects IgM and IgG antibodies directed against SARS-CoV-2. As our aim is to evaluate the efficacy of Covid-19 rapid tests and of serological assays in real-life settings, we designed a research protocol aimed to establish how to use correctly these diagnostics, taking into account the different possible clinical and epidemiological scenarios