Investigation of in vitro effects of ethephon and chlorpyrifos, either alone or in combination, on rat intestinal muscle contraction

A range of pesticides is widely used in pest management and the chances of exposure to multiple organophosphorus (OP) compounds simultaneously are high, especially from dietary and other sources. Although health hazards of individual OP insecticides have been relatively well characterized, there is lesser information on the interactive toxicity of multiple OP insecticides. The aim of this study is to elicit the possible interactions in case combined exposure of an OP pesticide chlorpyrifos (CPF) and a plant growth regulator ethephon (ETF) which are used worldwide. The ileum segments of 3 months old Wistar Albino male rats were used in isolated organ bath containing Tyrode solution. ETF and CPF were incubated (10−7 M concentration) separately or in combination with each other to ileum and their effects on acetylcholine-induced contractions were studied. The data obtained from this study show that, single and combined exposure to the agents caused agonistic interactions with regard to potency of ACh whereas they caused a decrease on Emax value of ACh. These findings suggest that exposure to these agents which have direct and indirect cholinergic effects, may cause developing clinical responses with small doses and earlier but the extent of toxicity will be lower

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes

A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane

BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure, Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Plasmid macro-evolution: selection of deletions during adaptation in a nutrient-limited environment

Under conditions where plasmid-carriage is deleterious to the cell, evolutionary changes may be expected which result in an attenuation of the deleterious effect of the plasmid. During long-term growth in glucose-limited continuous culture, initiated with a single clone of Escherichia coli containing a derivative of the plasmid pBR322, a structural change arose in the plasmid and predominated in the plasmid-containing sector of the population. This variant possessed a 2.25 kb deletion encompassing the tetracycline resistance operon as well as a region of about 1.5 kb upstream from this operon. Competition experiments involving strains carrying the plasmid with the spontancous deletion, and strains carrying plasmids with artificially constructed deletions, revealed that deletion of this region of the plasmid, involving loss of tetracycline resistance, resulted in an increment in fitness of between 10 and 20%. From the magnitude of the growth advantage, we conclude that the attenuation of the deleterious effect of the plasmid was mainly due to a reduction in the plasmid mediated interference in the metabolism of the cell caused by a deletion of the tetracycline resistance gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42797/1/10709_2004_Article_BF00127247.pd

Firing the climate canon: a literary critique of the genre of climate change

This article makes the case for more climate change, where climate change refers to the prevailing ideologies and frameworks that inform our understanding of environmental change in the first place. It reviews the mainstream literature in popular science writing, fiction and poetry from the point of view of a political frame analysis of climate change, to demonstrate how a certain understanding of climate change maps onto conventions of literary genre. This understanding, and associated literature, are critiqued on the basis of their continued attachment to dualistic and teleological narratives of human mastery and progress, such as to make the case for a literature which offers something radically other. The current political context, not least Donald Trump’s victory and Brexit, are cited as evidence of the contemporary importance of alternatives to the establishment approach to climate mitigation than either denial or scepticism – in both literature, and more broadly