Uses and Misuses of Agricultural Pesticides in Africa: Neglected Public Health Threats for Workers and Population

Pesticides are use in agriculture for their capacity to reduce pest and protect foods. Since their introduction in Africa by colonial masters, the use of these chemicals is constantly growing. Herbicides and insecticides are the two dominant categories. Although they are used in small quantities by farmers who own small exploitation, the frequency of their use, as well as overuses and misuses, constitutes serious factors of exposure and health risks. Farm workers are more vulnerable to occupational effects from pesticide inhalation and skin contacts. Failure to wear protective equipment and observe good agricultural practices explained health symptoms that are frequently experienced: eye and skin irritation, nausea, vomiting, and headache. Population is subject to chronic health effects due to repeated dietary intake of pesticides. Most consumed staple foods on the continent (cereals, vegetables, and fruits) have been found to be contaminated by one or multiple residues of pesticides. The level of residues is often higher than regulatory limits. Organization of surveillance programs to monitor concentration of pesticide residues remains inexistent in most countries, same for toxicovigilance systems to documented poisoning cases. Current data underline the need to carry out pesticide health risk assessment in order to appreciate the threats they pose for public health

Modulation of Ion Transport Across Rat Distal Colon by Cysteine

The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionine-β-synthase and cystathionine-γ-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and β-cyano-L-alanine, i.e., inhibitors of H2S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e., an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl− and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl− secretion, whereas Na cysteinate – after a transient inhibitory phase – activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K+ channels. Indeed, after preinhibition of basolateral K+ channels with tetrapentylammonium or Ba2+, the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H2S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H2S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H2S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter

Hypoxia/Reoxygenation Effects on Ion Transport across Rat Colonic Epithelium

Ischemia causes severe damage in the gastrointestinal tract. Therefore, it is interesting to study how the barrier and transport functions of intestinal epithelium change under hypoxia and subsequent reoxygenation. For this purpose we simulated hypoxia and reoxygenation on mucosa-submucosa preparations from rat distal colon in Ussing chambers and on isolated crypts. Hypoxia (N2 gassing for 15 min) induced a triphasic change in short-circuit current (Isc): a transient decrease, an increase and finally a long-lasting fall below the initial baseline. During the subsequent reoxygenation phase, Isc slightly rose to values above the initial baseline. Tissue conductance (Gt) showed a biphasic increase during both the hypoxia and the reoxygenation phases. Omission of Cl- or preincubation of the tissue with transport inhibitors revealed that the observed changes in Isc represented changes in Cl- secretion. The radical scavenger trolox C reduced the Isc response during hypoxia, but failed to prevent the rise of Isc during reoxygenation. All changes in Isc were Ca2+-dependent. Fura-2 experiments at loaded isolated colonic crypts revealed a slow increase of the cytosolic Ca2+ concentration during hypoxia and the reoxygenation phase, mainly caused by an influx of extracellular Ca2+. Surprisingly, no changes could be detected in the fluorescence of the superoxide anion-sensitive dye mitosox or the thiol-sensitive dye thiol tracker, suggesting a relative high capacity of the colonic epithelium (with its low O2 partial pressure even under physiological conditions) to deal with enhanced radical production during hypoxia/reoxygenation

The Rabl configuration limits topological entanglement of chromosomes in budding yeast.

The three dimensional organization of genomes remains mostly unknown due to their high degree of condensation. Biophysical studies predict that condensation promotes the topological entanglement of chromatin fibers and the inhibition of function. How organisms balance between functionally active genomes and a high degree of condensation remains to be determined. Here we hypothesize that the Rabl configuration, characterized by the attachment of centromeres and telomeres to the nuclear envelope, helps to reduce the topological entanglement of chromosomes. To test this hypothesis we developed a novel method to quantify chromosome entanglement complexity in 3D reconstructions obtained from Chromosome Conformation Capture (CCC) data. Applying this method to published data of the yeast genome, we show that computational models implementing the attachment of telomeres or centromeres alone are not sufficient to obtain the reduced entanglement complexity observed in 3D reconstructions. It is only when the centromeres and telomeres are attached to the nuclear envelope (i.e. the Rabl configuration) that the complexity of entanglement of the genome is comparable to that of the 3D reconstructions. We therefore suggest that the Rabl configuration is an essential player in the simplification of the entanglement of chromatin fibers

Mechanisms Associated to Nitroxyl (HNO)-Induced Relaxation in the Intestinal Smooth Muscle

The pharmacological properties of nitroxyl (HNO) donors in the gastrointestinal tract are unknown. We investigated the properties of this molecule in the regulation of gastrointestinal contractility focusing on its possible interaction with other gaseous signaling molecules such as NO and HS. Organ bath, Ca 2+ imaging, and microelectrode recordings were performed on rat intestinal samples, using Angeli's salt as HNO donor. Angeli's salt caused a concentration-dependent relaxation of longitudinal or circular muscle strips of the ileum and the proximal colon. This relaxation was strongly inhibited by the Rho-kinase inhibitor Y-27632 (10 μM), by the reducing agent DTT or by the inhibitor of soluble guanylate cyclase (sGC) ODQ (10 μM) alone or in combination with the inhibitors of the endogenous synthesis of HS β-cyano--alanine (5 mM) and amino-oxyacetate (5 mM). Preventing endogenous synthesis of NO by the NO synthase inhibitor L-NAME (200 μM) did not affect the relaxation induced by HNO. HNO induced an increase in cytosolic Ca 2+ concentration in colonic myocytes. It also elicited myocyte membrane hyperpolarization that amounted to −10.6 ± 1.1 mV. ODQ (10 μM) and Apamin (1 μM), a selective inhibitor of small conductance Ca 2+ -activated K + channels (SKca), strongly antagonized this effect. We conclude that HNO relaxes the gastrointestinal tract musculature by hyperpolarizing myocytes via activation of the sGC/cGMP pathway similarly to NO, not only inhibiting the RhoK and activating MLCP as do both NO and HS but also increasing cytosolic Ca 2+ for activation of SK contributing to hyperpolarization

Effects of H2O2 at rat myenteric neurones in culture

Oxidants, produced e.g. during inflammation, alter gastrointestinal functions finally leading to diarrhea and/or tissue damage. There is only scarce information about the action of oxidants on enteric neurones, which play a central role in the regulation of many gastrointestinal processes. Therefore, the effect of an oxidant, H2O2, on cultured rat myenteric neurones was studied with the whole-cell patch-clamp and imaging (fura-2) techniques. H2O2 (5 mmol/l) induced an increase in the cytosolic Ca2+ concentration. Both an intracellular release via IP3 and ryanodine receptors as well as a Gd3+-sensitive Ca2+ influx contributed to this response. Measurement of the membrane potential revealed that the neuronal membrane hyperpolarized by 11.3 ± 0.8 mV (n = 30) in the presence of H2O2. Inhibition of Ca2+-dependent K+ channels by the BK-specific inhibitor paxilline (10 µmol/l) or by 100 µmol/l TPA (a broad inhibitor of Ca2+-dependent K+ channels) prevented this hyperpolarization. This indicates that the increase in the cytosolic Ca2+ concentration likely activates BK channels, which carry an outward K+ current to hyperpolarize the neuronal membrane. Voltage-clamp experiments revealed a second action of the oxidant, i.e. a strong inhibition of the fast Na+ current responsible for the generation of action potentials. This effect seemed to be mediated by the hydroxyl radical (•OH), as Fe2+ (100 µmol/l), which leads to the generation of this radical from H2O2 via the Fenton reaction, strongly potentiated the action of an ineffective concentration (100 µmol/l) of the oxidant. Inhibition of protein phosphorylation by staurosporine (1 µmol/l), a protein kinase inhibitor, prevented the effect of a subsequent administration of the oxidant. Vice versa, the protein phosphatase (PP) inhibitor calyculin A (100 nmol/l) strongly reduced the inhibition of Na+ current by H2O2. This effect was mimicked by the PP2A specific inhibitor endothall (100 nmol/l), whereas the PP1 blocker tautomycin (3 nmol/l) was less effective. However, none of the inhibitors used was able to significantly change the basal amplitude of the inward sodium current. Changes appeared only after administration of the oxidant, meaning that a preconditioning of the channels or regulatory proteins, e.g. an oxidation of thiol groups by H2O2, is necessary for this action. This was confirmed by the observation that in the presence of the reduced form of GSH (3 mmol/l), H2O2 was unable to inhibit inward sodium currents. These results suggest that H2O2 might act via a shift of the equilibrium between protein phosphorylation and dephosphorylation involved in the regulation of Na+ currents in rat myenteric neurones. The consequence is an inhibition of the sodium currents responsible for the generation of action potentials. Together with the hyperpolarization, H2O2 should likely reduce the ongoing inhibitory tone of the ENS on the gastrointestinal muscle, which might result in a hypercontractility leading to diarrhea.Oxidantien, die z.B. während Entzündungsprozessen entstehen, beeinflussen Funktionen des Magen-Darm-Trakts, was im Endeffekt zu Durchfall und/oder Gewebeschäden führt. Es gibt nur wenige Daten über die Wirkung von Oxidantien an enteralen Neuronen, welche eine zentrale Rolle bei der Regulation vieler gastrointestinaler Prozesse spielen. Daher wurde mit der Hilfe von Whole-cell Patch-Clamp- und Imaging (Fura-2)-Techniken die Wirkung des Oxidationsmittels H2O2 an kultivierten myenterischen Neuronen aus der Ratte untersucht. Das Oxidationsmittel H2O2 (5 mmol/1) löste einen Anstieg der zytosolischen Ca2+ Konzentration aus. Sowohl eine intrazelluläre Freisetzung von Ca2+ über IP3- und Ryanodin-Rezeptoren als auch ein Gd3+-sensitiver Einstrom von Ca2+ sind an dieser Antwort beteiligt. Messung des Membranpotentials ergab, dass die Membran der myenterischen Neurone in Gegenwart von H2O2 um 11,3 ± 0,8 mV (n = 30) hyperpolarisierte. Die Hemmung von Ca2+-abhängigen K+-Kanälen durch den BK-spezifischen Inhibitor Paxillin (10 µmol/l) oder durch 100 µmol/l TPA (ein breit wirkender Blocker Ca2+-abhängiger K+-Kanäle) verhinderte diese Hyperpolarisation. Dies deutet darauf hin, dass der Anstieg der zytosolischen Ca2+ Konzentration wahrscheinlich BK-Kanäle aktiviert: diese Kanäle erlauben einen K+-Auswärtsstrom, der die Hyperpolarization bedingt. Spannungsklemm-Experimente deckten eine zweite Wirkung des Oxidationsmittels auf: Eine starke Hemmung des schnellen Na+-Stromes, der für die Erzeugung der Aktionspotentiale zuständig ist. Dieser Effekt scheint von dem Hydroxyl-Radikal (•OH) vermittelt zu sein, denn Fe2+ (100 µmol/l), das zur Erzeugung dieses Radikals aus H2O2 über die Fenton-Reaktion führt, potenzierte die Wirkung einer unwirksamen Konzentration (100 µmol/l) an H2O2. Die Hemmung der Protein-Phosphorylierung durch Staurosporin (1 µmol/l), einen Proteinkinase-Blocker, verhinderte die Wirkung des Oxidationsmittels. Umgekehrt reduzierte auch der Proteinphosphatase (PP)-Blocker Calyculin A (100 nmol/l) stark die von H2O2 induzierte Hemmung des Na+-Stromes. Dieser Effekt wurde durch den PP2A spezifischen Hemmer Endothall (100 nmol/l) nachgeahmt, während der PP1-Blocker Tautomycin (3 nmol/l) weniger wirksam war. Allerdings war keiner der verwendeten Inhibitoren in der Lage, die basale Amplitude des Na+-Einwärtsstroms zu beeinflussen. Änderungen erschienen erst nach der Zugabe des Oxidationsmittels, was bedeutet, dass eine Vorkonditionierung der Kanäle oder regulatorischer Proteine, wie zum Beispiel eine H2O2-bedingte Oxidation von Thiolgruppen, für diese Aktion notwendig ist. Dies wurde durch die Beobachtung, dass in Anwesenheit der reduzierten Form von GSH (3 mmol/l) H2O2 nicht in der Lage war, Na+-Einswärtsströme hemmen, untermauert. Diese Ergebnisse lassen vermuten, dass Oxidantien durch Veränderungen am enterischen Nervensystem Änderungen der gastrointestinalen Motilität auslösen können

Mechanical Performance Of Warm Mix Asphalt-Treated Bases Incorporating Recycled Asphalt Pavement

DissertationThe depletion of natural resources, the elevated cost related to the construction and the rehabilitation of pavements, the progressive change of the climate, the high heat and harmful gas emission into the atmosphere are the concerns engineers, academics, scientists and politicians have been addressing. They are co-operating toward finding efficient solutions to mitigate these global issues. Thus, the advent of Warm Mix Asphalt (WMA) incorporating Recycled Asphalt Pavement (RAP) as a long-term solution to partially or totally participating in remediating the problem of global warming, climate change and the preservation of environmental resources has gained prominent interest in certain European countries and Asia, North America and most recently in South Africa. This thesis, therefore, aims to investigate the performances of the control Hot Mix Asphalt (HMA) technology and the WMA incorporating RAP at 15% and 30% through laboratory experiment and numerical modelling. Consequently, the laboratory studies that involve the mix designs, the production and the testing of asphalt specimens were achieved following both the South African and the international standards. The RAP used at 15% and 30% in the WMA contains 0.8 % of 50/70 grade bitumen. The virgin aggregate called dolerite and the fillers used in the asphalt mixture were obtained at the Lafarge Olivehill Crushers site in Bloemfontein. The Sasobit, as well as the 50/70 grade bitumen binder, were collected in Sasolburg. The numerical simulation of the WMA – 15%RAP, the WMA – 30%RAP and the control HMA was achieved through the Finite Element Method (FEM) in the Abaqus computer program and the Layered Elastic Analysis (LEA) in mePADS. The Linear Elastic Analysis (LEA) was adopted not only to validate the results found in FEM but most of all, to justify the preference of FEM over the LEA. The numerical simulation WMA – 15%RAP pavement structures, the WMA – 30%RAP pavement structures and the control HMA pavement structures was to analyse their mechanical responses under repeated loading. The results of the laboratory experiment show that: the control HMA exhibits lower rutting performance than the WMA –15%RAP and WMA – 30%RAP; the control HMA exhibits lower fatigue cracking performance than the WMA –15%RAP and WMA – 30%RAP; the control HMA exhibits lower ITS (stiffness) performance when compared tothe WMA – 15%RAP the WMA – 30%RAP; the control HMA exhibits close Marshall Stability and Flow performance tothe WMA – 15%RAP the WMA – 30%RAP. As far as numerical modelling is concerned, the results show that the control HMA pavement structures exhibit lower rutting and fatigue cracking performance when compared to the WMA – 15%RAP pavement structures the WMA – 30%RAP pavement structures. Overall, the WMA can successfully incorporate RAP at up to 30% and can be utilized for the new construction and the rehabilitation of low to medium-traffic volume roads

The Native Copper- and Zinc- Binding Protein Metallothionein Blocks Copper-Mediated Aβ Aggregation and Toxicity in Rat Cortical Neurons

Background: A major pathological hallmark of AD is the deposition of insoluble extracellular b-amyloid (Ab) plaques. There are compelling data suggesting that Ab aggregation is catalysed by reaction with the metals zinc and copper. Methodology/Principal Findings: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Ab1–40 and Ab1–42. This action of MT-2A appears to involve a metal-swap between Zn 7MT-2A and Cu(II)-Ab, since neither Cu 10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Ab aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Ab induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. Conclusions/Significance: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Ab aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Ab leaving a metal-free Ab that can readily bind metals again, we believe that MT-2A might represent a different therapeuti